COMPOSITIONS COMPRISING SMALL EXTRACELLULAR VESICLES DERIVED FROM UMBILICAL CORD BLOOD MONONUCLEAR CELLS WITH ANTI-INFLAMMATORY AND IMMUNOMODULATORY PROPERTIES AND PROCESS FOR OBTAINING THEM
20210369789 · 2021-12-02
Inventors
Cpc classification
C12N2320/32
CHEMISTRY; METALLURGY
A61P29/00
HUMAN NECESSITIES
B01D15/34
PERFORMING OPERATIONS; TRANSPORTING
A61K35/51
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
C12N5/0037
CHEMISTRY; METALLURGY
International classification
A61K35/51
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
B01D15/34
PERFORMING OPERATIONS; TRANSPORTING
C12N5/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a process for isolating Small Extracellular Vesicles secreted by umbilical cord blood mononuclear cells (UCBMNCs) and compositions comprising said Small Extracellular Vesicles, which are useful to be applied to autoimmune diseases therapeutics or prophylactics and/or cosmetic purposes.
The proposed process for isolating UCBMNCs Small Extracellular Vesicles comprises three main steps: i) a first step of sequential centrifugation, ii) a second step of microfiltration combined with ultrafiltration (UF), and iii) a third step of size exclusion chromatography (SEC) and aims to achieve highly pure Small Extracellular Vesicles and in a higher yield.
The SEVs compositions comprise specific type of proteins, RNA and lipids, that enables them to be very effective when applied to inflammatory and autoimmune diseases therapeutics, such as psoriasis, lupus, atopic dermatitis, eczema, etc. and also to cosmetic or prophylactic compositions.
Therefore, the present invention lays in the technical domain of pharmaceuticals, medicine, cosmetics, research and development in cellular biology and appliances thereof.
Claims
1. A method (UF-SEC) for isolating Small Extracellular Vesicles (SEVs) secreted by umbilical cord blood mononuclear cells (UCBMNCs) comprising: i) a first step of sequential centrifugation, ii) a second step of microfiltration (MF) combined with ultrafiltration (UF), and iii) a third step of size exclusion chromatography (SEC), wherein said steps are performed as the following: a) Two rounds of sequential centrifugations performed first at low speed (200 g-400 g, 10-20 min), followed by average speed (1500 g-2500 g, 20-30 min), at a low temperature (4-10° C.), b) Filtration of the cleared supernatant of (a) by using sequentially a first filter having a pore size of 800 nm-450 nm and a second filter having a pore size of 250 nm-200 nm, c) Ultrafiltration of the solution obtained in (b) by using a molecule size cut-off of 100 KDa, at an adequate constant pressure, under stable stirring conditions of 100-200 rpm to achieve a solution of particles larger than 100 KDa and smaller than 200 nm, d) Concentration of the filtrate of (c) in up to 10× less the initial volume, e) Separation of SEVs from soluble proteins comprised in the solution of (d) by Size Exclusion Chromatography (SEC) using 280 nm absorbance signal for detection of SEVs, and f) Collection of SEVs fraction from the solution obtained in (e) at a stable temperature of 4 to 10° C., preferably of 3 to 5° C., when the absorbance of said solution at 280 nm increases in the first peak until the absorbance at 280 nm of that peak goes down to zero.
2. A process according to claim 1 further comprising a dilution step of the filtrate of step (b) with water, or a buffer solution with neutral pH, preferably a solution of phosphate buffer saline (PBS) of at least 1:1 and/or a washing step of the solution of (c) with water, or a buffer solution with neutral pH, preferably a PBS solution in a proportion of 1:4 respectively.
3. A process according to claim 1 wherein the UCBMNCs comprise lymphocytes, monocytes, neutrophils, eosinophils, and/or basophils.
4. A process according to claim 1 wherein the UCBMNCs consist essentially in lymphocytes, monocytes, neutrophils, eosinophils and/or basophils.
5. A process according to claim 1 wherein the UCBMNCs are cultured in a serum-free cell culture medium preferably supplemented with 10-1000 ng/ml of FMS-like tyrosine kinase-3 (Flt-3) and 10-1000 ng/ml stem-cell factor (STF) under ischemia conditions of 0.5% O.sub.2, and 5% CO.sub.2, at 37° C. for 18h.
6. A process according to claim 1 wherein the UCBMNCs are collected from at least 5 different blood donors and the obtained SEVs are pooled from said respective donors.
7. A process according to claim 1 further comprising a concentration step of SEVs fraction by using a filter with pore size of 10 KDa.
8. A process according to claim 1 further comprising a lyophilisation step.
9. A process according to claim 1 further comprising a SEVs modification step by enriching the SEVs compositions with addition of micro RNAs (miRNAs), palmitoylated tripeptides, peptides, DNA, small interference RNA (siRNA), growth factors, amino acids, sugars, lipid soluble molecules, fatty acids and their derivatives such as DHA, EPA, oleic acid, lipid modified molecules such as GPI anchored proteins or peptides, and/or other hydrophobic molecules, wherein said modification is done: a) before the initial steps of the isolation process, preferably during the cultivation of the UCBMNCs as described in claim 4, b) in a post-production phase of the isolation process after step h) as described in claim 1, or c) after the lyophilization step as described in claim 7.
10. A process according to claim 1 wherein the SEVs compositions are enriched by addition of selected miRNAs by direct hydration of the lyophilized SEVs compositions with a miRNA solution of 0.2-10 μM, preferably 0.25-5.0 μM, more preferably 0.5-1 μM.
11. A process according to claim 1 wherein the SEVs compositions are enriched by addition of palmitoylated tripeptides, such as Glycine-Histidine-Lysine tripeptide (GHK) or Lysine-Valine-Lysine tripeptide (KVK) preferably matrikines (MTK) by direct hydration with solutions of different concentrations varying in the range of 20 uM to 60 uM, during approximately 1 hour at 37° C.
12. A Small Extracellular Vesicles (SEVs) composition obtainable by the process as described in claim 1 wherein the SEVs consist in one or more bioactive molecules selected from the following (a) proteins, (b) miRMAs and (c) lipids: a) Proteins: TABLE-US-00012 UniProt Nr. ID Name 1 P0DOX2 IGA2 2 P0DOX7 IGK 3 P01871 IGHM 4 P01619 KV320 5 Q5VTE0 EF1A3 6 P01876 IGHA1 7 P04217 A1BG 8 P01023 A2M 9 P63261 ACTG1 10 P12814 ACTN1 11 P43652 AFM 12 P02765 AHSG 13 P02768 ALB 14 P04075 ALDOA 15 P02760 AMBP 16 P04083 ANXA1 17 P50995 ANXA11 18 P07355 ANXA2 19 P12429 ANXA3 20 P08758 ANXA5 21 P08133 ANXA6 22 P20073 ANXA7 23 O75882 ATRN 24 P25311 AZGP1 25 P30043 BLVRB 26 P02745 C1QA 27 P02746 C1QB 28 P02747 C1QC 29 P09871 C1S 30 P01024 C3 31 P04003 C4BPA 32 P02748 C9 33 P00918 CA2 34 P04040 CAT 35 Q08722 CD47 36 P08962 CD63 37 P23528 CFL1 38 P10909 CLU 39 P31146 CORO1A 40 P00450 CP 41 P01040 CSTA 42 P08311 CTSG 43 P81605 DCD 44 P59666 DEFA3 45 Q02413 DSG1 46 P15924 DSP 47 P60842 EIF4A1 48 P08246 ELANE 49 P06733 ENO1 50 P16452 EPB42 51 P21333 FLNA 52 P02751 FN1 53 P04406 GAPDH 54 P02774 GC 55 P13224 GP1BB 56 P06396 GSN 57 O15511 ARPC5 58 P46976 GYG1 59 P69905 HBA1 60 P68871 HBB 61 P02042 HBD 62 P69891 HBG1 63 P69892 HBG2 64 P02008 HBZ 65 P62805 HIST4H4 66 P00738 HP 67 P02790 HPX 68 Q86YZ3 HRNR 69 P11142 HSPA8 70 P08514 ITGA2B 71 P05106 ITGB3 72 P01591 JCHAIN 73 P14923 JUP 74 P01042 KNG1 75 P11279 LAMP1 76 P31025 LCN1 77 P07195 LDHB 78 Q08380 LGALS3BP 79 P02750 LRG1 80 P02788 LTF 81 P61626 LYZ 82 Q08431 MFGE8 83 P05164 MPO 84 P35579 MYH9 85 P60660 MYL6 86 Q6UX06 OLFM4 87 P07737 PFN1 88 P14618 PKM 89 P55058 PLTP 90 P02775 PPBP 91 P62937 PPIA 92 P32119 PRDX2 93 P30041 PRDX6 94 P05109 S100A8 95 P06702 S100A9 96 P29508 SERPINB3 97 P05155 SERPING1 98 P11166 SLC2A1 99 P02730 SLC4A1 100 P11277 SPTB 101 P30626 SRI 102 P27105 STOM 103 P02787 TF 104 P02786 TFRC 105 P07996 THBS1 106 Q9Y490 TLN1 107 P24821 TNC 108 P60174 TPI1 109 P62987 UBA52 110 P13611 VCAN 111 P18206 VCL 112 P31946 YWHAB 113 P63104 YWHAZ 114 PODOX5 IGG1 115 P16157 ANK1 116 P0C0L5 C4B 117 P31944 CASP14 118 Q16778 HIST2H2BE 119 P01859 IGHG2 120 Q05707 COL14A1 121 Q01518 CAP1 122 P02549 SPTA1 123 P16403 HIST1H1C 124 P00736 C1R 125 P0DOY2 IGLC2 126 P12273 PIP 127 Q96P63 SERPINB12 128 P07478 PRSS2 129 P02649 APOE 130 Q14254 FLOT2 131 Q08188 TGM3 132 P67936 TPM4 133 Q06830 PRDX1 134 O43866 CD5L 135 P08238 HSP90AB1 136 Q9NZD4 AHSP 137 P01768 HV330 138 Q71DI3 HIST2H3C 139 P40926 MDH2 140 P19338 NCL 141 Q93077 HIST1H2AC 142 P01031 C5 143 P20618 PSMB1 144 O00299 CLIC1 145 P09105 HBQ1 146 Q9UL46 PSME2 147 Q86TJ2 TADA2B 148 Q16610 ECM1 149 P60900 PSMA6 150 P53396 ACLY 151 P37802 TAGLN2 152 P35754 GLRX 153 P29972 AQP1 154 P28072 PSMB6 155 P28066 PSMA5 156 P06331 HV434 157 O75635 SERPINB7 158 O75368 SH3BGRL 159 P59190 RAB15 160 Q08554 DSC1 161 P01743 HV146 162 P80188 LCN2 163 P05089 ARG1 164 O14818 PSMA7 165 Q6ZVX7 NCCRP1 166 P19652 ORM2 167 P48059 LIMS1 168 P29353 SHC1 169 P10599 TXN 170 Q5T749 KPRP more preferably one or more proteins selected from the group of ANXA2, ANK1, CD63, CD81, CD9, CD15, more preferably wherein said proteins are present in the SEVs compositions in the amounts of: CD81≥1%, CD9≥1%, CD63≥40%, CD15≥20% positive events as measured by flow cytometry of SEVs coupled to microbeads, or quantity of CD63≥5 μg/mL or ANXA2≥0.3 ng/mL in the purified SEVs, as measured by ELISA; b) miRNAs: TABLE-US-00013 hsa-let-7a-5p hsa-miR-15a-5p hsa-miR-2l-5p hsa-let-7f-5p hsa-miR-15b-5p hsa-miR-223-3p hsa-let-7g-5p hsa-miR-16-5p hsa-miR-23a-3p hsa-miR-103a-3p hsa-miR-17-5p hsa-miR-26a-5p hsa-miR-106b-5p hsa-miR-191-5p hsa-miR-26b-5p hsa-miR-142-3p hsa-miR-19a-3p hsa-miR-29b-3p hsa-miR-146a-5p hsa-miR-19b-3p hsa-miR-30d-5p hsa-miR-150-5p hsa-miR-20a-5p hsa-miR-451a hsa-let-7b-5p hsa-miR-185-5p hsa-miR-30b-5p hsa-let-7c-5p hsa-miR-18a-5p hsa-miR-3184-3p hsa-let-7i-5p hsa-miR-205-5p hsa-miR-376c-3p hsa-miR-130a-3p hsa-miR-221-3p hsa-miR-486-5p hsa-miR-144-3p hsa-miR-22-3p hsa-miR-92a-3p hsa-miR-144-5p hsa-miR-27a-3p hsa-miR-93-5p hsa-miR-181a-5p hsa-miR-27b-3p preferably comprising one or more of the following miRNAs: hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7i-5p, hsa-miR-130a-3p, hsa-miR-144-3p, hsa-miR-144-5p, hsa-miR-181a-5p, hsa-miR-185-5p, hsa-miR-18a-5p, hsa-miR-205-5p, hsa-miR-221-3p, hsa-miR-22-3p, hsa-miR-27a-3p, hsa-miR-27b-3p, hsa-miR-30b-5p, hsa-miR-3184-3p, hsa-miR-376c-3p, hsa-miR-486-5p, hsa-miR-92a-3p and hsa-miR-93-5p, more preferably miR-150-5p, miR-223-3p, miR-16-5p, miR-142-3p, miR-19b, and more preferably wherein said miRNAs are present in the SEVs composition in the respective amounts of: miR-150-5p>1.3 μg/10{circumflex over ( )}9 part, miR-223-3p>1.2 μg/10{circumflex over ( )}9 part, miR-16-5p>0.5 μg/10{circumflex over ( )}9 part, miR-142-3p>0.4 μg/10{circumflex over ( )}9 part, miR-19b>0.2 μg/10{circumflex over ( )}9 part, as measured by direct quantification, and c) lipids: cholesteryl esters (CE), Diacylglycerides (DAG), phosphatidic acid (PA), phosphatidylcholines (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserines (PS), Sphingomyelin (SPM), Triacylglycerides (TAG), preferably phosphatidylcholines (PC), phosphatidylserines (PS) and Sphingomyelin (SPM), and more preferably wherein said lipids are present in the SEVs composition in a minimum percentage, respective to the total lipid amount of: CE≥0.05±0.05, DAG≥1.3±0.4, PA≥3.18±0.6, PC≥30.9±3.3, PE≥13.0±0.5, PG≥0.25±0.1, PI≥3.8±0.3, PS≥29.4±3.7, SPM≥18.0±1.6, TAG≥0.074±0.13.
13. A Small Extracellular Vesicles (SEVs) composition according to claim 12 consisting in one or more bioactive molecules selected from the following (a) proteins, (b) miRNAs and (c) lipids as described in claim 12.
14. A Small Extracellular Vesicles (SEVs) composition according to claim 13 wherein: a) the proteins present in said compositions consist in combinations of the presented proteins, as in claim 13, preferably in combinations of proteins selected from the group of ANXA2, ANK1, CD63, CD81, CD9, CD15, more preferably wherein the following proteins are present in the SEVs in the amounts of: CD81≥1%, CD9≥1%, CD63≥40%, CD15≥20% positive events as measured by flow cytometry of SEVs coupled to microbeads, or quantity of CD63≥5 μg/mL or ANXA2≥0.3 ηg/mL in the purified SEVs, as measured by ELISA; b) the miRNA present in said compositions consist in combinations of the presented miRNA, as in claim 13, preferably in combinations of miRNAs selected from the following: miR-150-5p>1.3 μg/10{circumflex over ( )}9 part, miR-223-3p>1.2 μg/10{circumflex over ( )}9 part, miR-16-5p>0.5 μg/10{circumflex over ( )}9 part, miR-142-3p>0.4 μg/10{circumflex over ( )}9 part, miR-19b>0.2 μg/10{circumflex over ( )}9 part, as measured by direct quantification, and c) the lipids present in said compositions consist in combinations of the presented lipids, as in claim 13, preferably in combinations of lipids selected from CE, Diacylglycerides (DAG), PA, phosphatidylcholines (PC), PE, PG, PI, phosphatidylserines (PS), Sphingomyelin (SPM), Triacylglycerides (TAG), more preferably phosphatidylcholines (PC), phosphatidylserines (PS) and Sphingomyelin (SPM), and more preferably in the following respective percentages, respective to the total lipid amount of: CE≥0.05_±0.05, DAG≥1.3±0.4, PA≥3.18±0.6, PC>30.9±3.3, PE≥13.0±0.5, PG≥0.25±0.1, PI≥3.8±0.3, PS≥29.4±3.7, SPM≥18.0±1.6, TAG≥0.074±0.13.
15. A Small Extracellular Vesicles (SEVs) composition according to claim 14 consisting in the following combination of proteins, miRNAs and lipids: Proteins: ANXA2, CD63, CD81, CD9 and CD15, RNAs: preferably miR-150-5p, miR-223-3p, miR-16-5p, miR-142-3p, miR-19b, and Lipids: CE≥0.05±0.05, DAG≥1.3±0.4, PA≥3.18±0.6, PC≥30.9±3.3, PE≥13.0±0.5, PG≥0.25±0.1, PI≥3.8±0.3, PS≥29.4±3.7, SPM≥18.0±1.6, TAG≥0.074±0.13.
16. A Small Extracellular Vesicles (SEVs) composition according to claim 12 wherein the SEVs are further enriched by addition of miRNAs, palmitoylated tripeptides, peptides, DNA, siRNA, growth factors, amino acids, sugars, lipid soluble molecules, fatty acids and its derivatives such as DHA, EPA, Oleic acid, lipid modified molecules such as GPI anchored proteins or peptides, and/or other hydrophobic molecules, preferably SEVs are enriched miRNAs, or palmitoylated tripeptides, preferably the SEVs are enriched by addition of one or more miRNAs selected from a group consisting of: hsa-miR-150-5p, hsa-miR-16-5p, hsa-miR-142-3p, hsa-miR-223-3p, hsa-let-7 g-5p, hsa-miR-21-5p, hsa-let-7f-5p, hsa-miR-19b-3p, hsa-let-7a-5p, hsa-miR-26a-1-5p, hsa-miR-20a-5p, hsa-miR-181a-5p, hsa-miR-451a, hsa-miR-23a-3p, hsa-miR-342-3p, hsa-miR-191-5p, hsa-miR-103a-3p, hsa-miR-15a-5p, hsa-miR-142-5p, hsa-miR-146a-5p, hsa-miR-19a-3p, hsa-miR-15b-5p, hsa-miR-26b-5p, hsa-miR-30d-5p, hsa-miR-146b-5p, hsa-miR-106b-5p, hsa-miR-29a-3p, hsa-miR-17-5p, hsa-miR-29b-3p, and hsa-miR-101-3p, more preferably the SEVs are enriched with one or more miRNAs selected from a group consisting of: hsa-miR-181a-5p, hsa-miR-451a, hsa-miR-103a-3p, hsa-miR-15a-5p, hsa-miR-19a-3p, hsa-miR-15b-5p, hsa-miR-26b-5p, hsa-miR-30d-5p, hsa-miR-146b-5p, hsa-miR-106b-5p, hsa-miR-29a-3p, hsa-miR-17-5p, hsa-miR-29b-3p, and hsa-miR-101-3p, most preferably the SEVs are enriched with miRNAs hsa-miR-150-5p, thereby increasing the concentration of miRNAs in the SEVs composition.
17. (canceled)
18. A pharmaceutical composition comprising a Small Extracellular Vesicles (SEVs) composition as described in claim 12 in the form of a liquid, a powder, a spray, a cream, a gel or hydrogel, for use as cosmetic, as a medicament or as prophylactic agent.
19. (canceled)
20. A pharmaceutical composition according to claim 18 for use in inflammatory, autoimmune or other immune-system diseases therapy and/or prophylaxis, in particular: a) for use in autoimmune diseases associated to inflammation diseases therapy and/or prophylaxis, b) for use in autoimmune diseases with skin manifestations such as Psoriasis, Systemic Lupus Erythematosus (SLE), Vitiligo, scleroderma, alopecia, pemphigus, atopic dermatitis or eczema diseases therapy and/or prophylaxis, or c) for use in local or systemic inflammatory diseases, with or without autoimmune cause, such as inflammatory bowel disease, atherosclerosis, ulcerative colitis, Crohn' disease, hepatitis or transplant rejection diseases therapy and/or prophylaxis, d) for use in tissue repair diseases therapy and/or prophylaxis, e) for use in skin tissue repair diseases therapy and/or prophylaxis, f) for use in skin open wounds, such as an incision, a laceration, a tear, an abrasion, an avulsion or a surgical wound diseases therapy and/or prophylaxis, g) for use in skin burn therapy, such as a thermal burn, a chemical burn or a radiation burn diseases therapy and/or prophylaxis, h) for use in wounds such as arterial ulcers, venous ulcers, diabetic ulcers and pressure ulcers, post-surgical ulcer, traumatic ulcer, mouth ulcer, diabetic foot ulcer or corneal ulcer diseases therapy and/or prophylaxis.
21-30. (canceled)
31. A process according to claim 1 wherein SEVs compositions are enriched by addition of biologically active molecules, namely miRNAs or lipid-soluble molecules comprising the following steps: a) Providing a dehydrated or lyophilized powder composition of SEVs to be enriched, b) Direct hydration of the SEVs composition with a solution of selected biologically active molecules composition by: (i) Incubation of a SEVs composition having 1×10{circumflex over ( )}9 to 1×10{circumflex over ( )}11 particles/mL, preferably 1×10{circumflex over ( )}11, with the same volume of a solution of a selected miRNA, said miRNA solution comprising an amount of miRNA of 0.2-10 μM, preferably 0.25-5.0 μM, more preferably 0.5-1 μM in water, or a buffer solution with neutral pH, such as PBS, in 3 cycles at approximately 37° C. for around 2 min, each cycle followed by vortex, or by (ii) Incubation of a SEVs composition having 1×10{circumflex over ( )}9 to 1×10{circumflex over ( )}11 particles/mL, preferably 1×10{circumflex over ( )}11, with the same volume of a solution of a selected lipid-soluble molecule solution, said lipid-soluble molecule is preferably a palmitoylated tripeptide, more preferably matrikine (MTK), comprising concentrations between 10-60 uM in water, or a buffer solution with neutral pH, such as PBS, and optionally further comprising a filtration step performed with a 10-100 kDa, preferably 20-70 kDa, more preferably 40-50 kDa molecule cut-off followed by three rounds of washing with water or a buffer solution with neutral pH, preferably with PBS, and resuspension in a buffer solution with neutral pH, preferably PBS, by centrifugation at high speed, preferably 14000 g at 4° C., 4-10 min.
Description
DESCRIPTION OF THE FIGURES
[0031]
[0032]
[0039]
[0043] The error bars represent the SEM. The statistical test performed was a One-Way ANOVA with: ****p<0.0001.
[0044]
B.1—Fluorescence spectra of the solution containing a fluorescent miRNA mimic with (gray) and without (black) SEVs.
B.2—Fluorescence of the top compartment (Filter) after 1 centrifugation of a solution containing 1 uM of miR and no SEVs.
B.3, B.4—Fluorescence of the top compartment (Filter) after 3 centrifugation steps of a solution containing 1 uM of fluorescence miR and 1×10.sup.11 part/mL SEVs. After 3 centrifugations the remaining fluorescence corresponds to miR that was incorporated in the SEVs.
[0045]
A. Schematic workflow for the modification of the SEVs with MTK.
B.1—Absorption spectra of the solution containing a fluorescent MTK SEVs.
B.2—Fluorescence of the MTK modified SEVs.
[0046]
[0050] The error bars represent the SEM. The statistical test performed was a One-Way ANOVA with: ** for p<0.01 and *** for p<0.001.
[0051]
[0055] The error bars represent the SEM. The statistical test performed was a One-Way ANOVA with: ** for p<0.01 and *** for p<0.001.
[0056]
[0057] Proliferation index measured by CFSE intensity of: [0058] A. overall T cell proliferation (CD3+ cells); [0059] B. helper T cells (CD3+CD4+); [0060] C. cytotoxic T cell subset (CD3+CD8+); [0061] D. Overall T cell viability. Internalization of 7-AAD was used to stain death cells.
[0062] Results are presented as mean±SEM (n=6 for SEVs and n=1 for PBMNCs donor). Statistical analysis was performed using one-way ANOVA. * for p<0.05; *** for p<0.001 and **** for p<0.0001.
[0063]
[0067]
[0071] Results are presented as mean±SEM (n=6 for SEVs and n=1 for PBMNCs donor). Statistical analysis was performed using one-way ANOVA. * for p<0.05; *** for p<0.001 and **** for p<0.0001.
[0072]
[0073]
[0078] Results are presented as mean±SEM (n=6 for SEVs and n=2 for PBMNCs donor). Statistical analysis was performed using one-way ANOVA. * for p<0.05; ** for p<0.01; *** for p<0.001 and **** for p<0.0001.
[0079]
A. human TNF-α secretion by PBMNCs;
B. human CCL20 secretion by PBMNCs.
[0080] Results are presented as mean±SEM (n=3 for SEVs and n=1 for PBMNCs donor). Statistical analysis was performed using one-way ANOVA. * for p<0.05; ** for p<0.01; *** for p<0.001 and **** for p<0.0001.
[0081]
[0088] Results are presented as mean±SEM (n=3 for SEVs and n=1 for PBMNCs donor). Statistical analysis was performed using one-way ANOVA. * for p<0.05; ** for p<0.01; *** for p<0.001 and **** for p<0.0001.
[0089]
[0090]
[0095] Results are presented as mean±SEM (n=3 for SEVs). Statistical analysis was performed using student t-test. * for p<0.05; ** for p<0.01.
[0096]
[0101]
[0104] Results are presented as mean±SEM (n=6). Statistical analysis was performed using one-way ANOVA. * for p<0.05; ** for p<0.01.
[0105]
[0108]
[0109] Full-thickness excisional wounds were performed on the skin of 16 chemically diabetes-induced C57BL/6 mice after 50 days of induction with STZ (50 mg/kg). Each mouse was randomly assigned to each treatment group: Inventive Method or Prior art Method. SEVs treatment was topically applied twice a day for 15 days. (A) Representative wounds micrographs of each condition at day 0, 3 and 10. Scale bar: 0.5 cm. (B) Arithmetic mean and SEM (n≥12) of the wound size percentage for all the mice, at each time point (every day). *p≤0.05, *p≤0.01, *p≤0.0001.
DESCRIPTION OF THE INVENTION
[0110] The present invention relates to a process for isolating Small Extracellular Vesicles secreted by umbilical cord blood mononuclear cells (UCBMNCs) and compositions comprising Small Extracellular Vesicles obtained by processing said cells, which are useful to be applied to autoimmune and/or immune-system diseases therapeutics or prophylactics and/or cosmetic purposes.
1. Process for Isolating Mononuclear Cells Umbilical Cord Blood Small Extracellular Vesicles
[0111] The process of the present invention can be applied to isolating Small Extracellular Vesicles secreted by umbilical cord blood mononuclear cells (UCBMNCs). This process aims to achieve highly pure Small Extracellular Vesicles and in a higher yield.
[0112] It comprises i) a first step of sequential centrifugation, ii) a second step of microfiltration combined with ultrafiltration (UF), and iii) a final step of size exclusion chromatography (SEC).
[0113] In general, the process of the invention may be performed according to the following:
1.1 Collection and Processing of the UCBMNCs
[0114] Human cord blood samples can be obtained from healthy volunteer donors after providing their informed consent. Blood cells are then isolated by gradient centrifugation, for example being automatically processed through an Automated Cord Blood Processing system. During centrifugation, component stratification and separation occurs, and cord blood components (buffy coat, plasma, red blood cells) can be directed into separate sterile bags.
[0115] In summary, red blood cells (RBC) are transferred to a separate sterile bag, the buffy coat, which contains the mononuclear cell (MNC) rich layer that can be delivered to a separate sterile freezing bag, while the plasma remains in the processing bag.
[0116] Said UCBMNCs comprise lymphocytes, monocytes, neutrophils, eosinophils, and/or basophils.
[0117] It is known in the art that this automated process overcomes the manual processing limitations such as technical variability between users and inefficiency in the processing time and cost. More importantly, it does not compromise the cell viability and potency.
1.2 UCB Cell Culture
[0118] Cells can be cultured for a minimum period of approximately 18h with or without ischemia. UCBMNCs are cultured in a serum-free cell culture medium preferably supplemented with 10-1000 ng/ml of FMS-like tyrosine kinase-3 (Flt-3) and 10-1000 ng/ml stem-cell factor (SCF) without ischemia or preferable under ischemia conditions, for example of 0.5% O.sub.2, and 5% CO.sub.2, at 37° C. for 18 h.
[0119] Small Extracellular Vesicles secreted by cells subjected to ischemic conditions (O.sub.2<2%) are more effective in inducing tissue repair (especially when damage is perpetuated by local ischemia) than Small Extracellular Vesicles secreted by cells when not exposed to ischemic conditions. This is especially because they are enriched in biomolecules that promote neovascularization. Therefore, in a preferred embodiment of the invention, cells are cultured under ischemic conditions.
1.3 SEVs Isolation
[0120] Small Extracellular Vesicles (SEVs) secreted by umbilical cord blood mononuclear cells (UCBMNCs), directly the buffy coat obtained from the UCB automatic processing, are purified (isolated) using a three-step process, according to the present invention, wherein i) the medium is cleared from large particles and cell debris using two rounds of sequential centrifugations; ii) the cleared supernatant is subjected to sequential micro (MF) and ultrafiltration (UF) techniques, to eliminate vesicles and other particles with more than 200 nm and less then 100 kDa in size; and iii) the final separation step with high performance exclusion chromatography (SEC), which further separates the isolated SEVs and eliminates small soluble proteins.
[0121] A preferred embodiment of the process of the invention is described in
[0122] Initially, the medium is cleared from large particles and cell debris using two rounds of sequential centrifugations typically performed first at low speed (200 g-400 g, 10-20 min), followed by average speed (1500 g-2500 g, 20-30 min), at a low temperature (4-10° C.), thus resulting in a cleared Conditioned Medium.
[0123] Afterwards, the collected Conditioned Medium is microfiltered either manually, or using automated systems such as including a peristaltic pump (<1 Bar) by performing a sequentially filtration using a first filter with a pore filter of 800 nm-450 nm followed by another filtration using a second smaller filter with pore filter of 250 nm-200 nm. This filtration step can also be performed by an apparatus combining both above mentioned filters.
[0124] This filtered solution is preferably then diluted with a phosphate buffer saline (PBS) in a ratio of at least 1:1.
[0125] The resulting solution is then subjected to an ultrafiltration step. This can be done by using a Vivacell® 250 system, under pressure of 2-4Bar, preferably 3-3,5Bar, and constant stirring conditions of 100-200 rpm. A tangential field flow system, using a molecule size cut-off of 100 KDa is also adequate for said purpose.
[0126] This result in a concentrated solution of particles bigger than 100 KDa and smaller than 200 nm, including SEVs.
[0127] The concentrated solution is then preferably washed 1 time with a PBS solution in a ratio of at least 1:4, respectively, and concentrated again to a minimal volume compatible with the following purification steps, preferably at least 10× less the initial volume.
[0128] The final isolation step uses high-performance size exclusion chromatography (SEC).
[0129] This step can be performed in a suitable equipment for SEC, such as a FPLC− Akta Avant Chromatographic system, or equivalent. A filtered PBS solution is used as elution buffer at a compatible flow rate, according to the SEC column used (0.5 mL/min in Superose 6 10/300 GL; 3 mL/min in XK 26/70).
[0130] UV absorbance was detected at three different wavelengths (220, 260 and 280 nm), while the 280 nm is used as the preferred signal for the SEVs detection.
[0131] Two individualized peaks can be observed in the chromatogram, and the SEVs are present in the first eluted peak (particles bigger in size). The second peak detected corresponds to smaller soluble proteins that are still present in the injected solution.
[0132] SEVs start to be collected when the absorbance at 280 nm increases in the first peak, and collection ends when the absorbance at 280 nm of that peak goes down to zero.
[0133] Accordingly, SEVs fraction corresponds to elution volumes between 8-10 ml in Superose GE 100/300 and to 100-120 ml in Superose GE XK 26/70 (
[0134] Analysis of the collected fractions by Nanoparticle Tracking Analysis (NTA) and measurement of Protein Concentration is used to confirm the presence of SEVs in the isolated samples. SEVs correspond to higher number of nanosized particles with low protein concentration, while contaminant soluble proteins of the second peak correspond to low number of nanosized particles with high protein concentration (
[0135] The SEVs isolated according to the optimized methodology (UF-SEC) of the invention have similar morphological characteristics when compared with SEVs isolated according to the described prior art process (UC).
[0136] SEVs were evaluated by FPLC techniques in function of their individual amounts and types in the resulting compositions, i.e. SEVs species, and it was observed that the contamination with soluble proteins is less than 20%, which is very similar to the prior art UC methodology (
[0137] Nanoparticle Tracking Analysis (NTA technique) is used to analyse the size distribution of the isolated SEVs. The profile of the curve is centred in sizes below 200 nm, and the average size obtained is close to 150 nm (
[0138] Presence of microvesicles (200-400 nm) in the isolated SEVs fractions is within 5-25% (
[0139] The yield in number of isolated SEVs is higher when using the optimized methodology (UF-SEC) of the invention, comparing to the prior art method (UC), independently of the normalization used: (i) per millilitre of conditioned medium used (
[0140] These results show that in respect to SEVs yield the process of the invention (UF-SEC) is superior than the process of the prior art (UC).
1.4 SEVs Pooling
[0141] Small Extracellular Vesicles are pooled from several blood donors and are characterized according to optimized techniques and standards. The pooling can be made from at least 5 different donors, using the cell culture conditioned medium, or after the purification of the Small Extracellular Vesicles. Using this strategy, it is possible to increase the homogeneity between the purified Small Extracellular Vesicles, without affecting their bioactive properties.
1.5 Lyophilisation
[0142] A lyophilisation step may be added to the general process in order to produce a storable product and/or to produce compositions that can be used in later occasions.
[0143] Lyophilisation is a dehydration process through the sublimation process. This controlled drying process removes the water, leaving the molecular structure of the product intact. Given that the desired final product can be a lyophilized formulation, tests were performed to access whether lyophilisation could affect the stability and biochemical/biophysical characteristics of the SEVs compositions or fractions. The lyophilisation can be performed for approximately 12-24h depending on the volume with a pressure<0.4 mBar and temperature of approximately −50° C.
[0144] In terms of morphological characteristics of the extracellular vesicle compositions after lyophilization, 5 pools were tested, each containing 5 different blood donors. It was observed that the impact of lyophilisation is negligible. Both the modal size and the concentration seem not to be affected by the lyophilisation procedure, indicating that they are maintained stable in the process and are well hydrated by our solvent. The relative expression of surface markers is also not significantly affected by the lyophilisation, being an indication that the surface properties are conserved throughout the procedure.
2. Method for Small Extracellular Vasicles (SEVs) Modification
[0145] Isolated Small Extracellular Vesicles (SEVs) can be modified to increase their bioactive efficiency.
[0146] These modifications may be done by pre-conditioning the secreting cells (UCBMNCs) with specific stimuli, or directly in the SEVs compositions after isolation with the process of the invention (UF-SEC).
[0147] In the present invention, a post-isolation modification is preferred. For this purpose, SEVs compositions obtained by the UF-SEC methodology are enriched with specific molecules, such as selected miRNAs, which are responsible or involved in the process of treating and/or preventing certain diseases, such as inflammatory and autoimmune diseases, or in cosmetic applications.
[0148] The obtained SEVs compositions that can be presented either in liquid or in powder form.
[0149] In a preferred embodiment of the invention, SEVs are modified with selected types of miRNA by direct hydration of the lyophilized powder composition of SEVs with a miRNA solution (
[0150] Lyophilized SEVs compositions having 1×10{circumflex over ( )}9-1×10{circumflex over ( )}11 particles/mL, preferably 1×10{circumflex over ( )}11, are directly rehydrated with the same volume of a miRNA solution of 0.2-10 uM, preferably 0.25-5.0 μM, more preferably 0.5-1 μM in water, or a buffer solution with neutral pH, such as PBS, and 3 cycles of incubation at 37° C. for 2 min are performed, each cycle followed by vortex.
[0151] Therefore, the method for SEVs modification with miRNA molecules comprise the following steps: [0152] a) Providing a powder composition of SEV to be modified; [0153] b) Direct hydration of the SEVs composition with a solution of selected miRNA comprising an amount of miRNA of 0.2-10 μM, preferably 0.25-5.0 μM, more preferably 0.5-1 μM in water, or a buffer solution with neutral pH, such as PBS, [0154] c) Incubation of the mixture in 3 cycles at approximately 37° C. for around 2 min, each cycle followed by vortex.
[0155] Optionally, a filtration step is added to the above method performed with a 10-100 kDa, preferably 20-70 kDa, more preferably 40-50 kDa molecule cut-off to separate the non-incorporated miRNA, and three rounds of washing with water or a buffer solution with neutral pH, preferably with PBS are performed, typically with resuspension in a buffer solution with neutral pH, preferably PBS, centrifugation at 14000 g at 4° C., 4 min. The modified SEVs can then be collected by a known method in the art.
[0156] Another approach for Small Extracellular Vesicles enrichment was tested with the incorporation of a palmitoylated tripeptide, such as matrikine (MTK) in the SEVs compositions (
[0157] Given that palmitoylated tripeptides, such as MTK, are lipid soluble molecules, the modification methodology as described above had to be adapted.
[0158] Therefore, the method for SEVs modification with palmitoylated tripeptides molecules comprise the following steps: [0159] a) Providing a powder composition of SEV to be modified; [0160] b) Direct hydration of the SEVs composition with a solution of selected palmitoylated tripeptides comprising concentrations between 10-60 uM in water, or a buffer solution with neutral pH, such as PBS; [0161] c) Incubation of the mixture for 1 h at 37° C.
[0162] The non-incorporated pal-tripeptide can be removed by a filtration step with a 10-100 kDa, preferably 20-70 kDa, more preferably 40-50 kDa molecule cut-off to separate the non-incorporated palmitoylated tripeptide, and three rounds of washing with PBS (resuspend in PBS, centrifuge at 14000 g at 4° C., 4 min), as described above.
3. Analysis of SEVs Modification
[0163] For accessing the validity of the described SEVs modification strategies, fluorescence miRNA and fluorescent MTK was incorporated into the SEVs powder composition.
[0164] SEVs were modified by direct hydration as described above with fluorescent miRNA solution. In the reference solution (containing the free incorporated fluorescent miRNA), after the first centrifugation the fluorescence was not retained by the filter (fluorescence in the top compartment goes to zero), whilst the fluorescence in the SEVs composition was maintained at about 20% of the initial value (FIGS. 4B3 and 4B4).
[0165] In the washing of the filtration step the fluorescent SEVs were recovered by centrifugation after each washing step. The fluorescence was maintained in 20% after the three washings, which demonstrates an efficient incorporation of the fluorescent miRNA into the SEVs. To further evaluate the efficacy of the modifications with miRNA, real-time PCR is used to evaluate the amount of miRNA before and after the modification strategy.
[0166] For validation of incorporation of palmitoylated tripeptides, similarly to the miRNA validation methodology, a fluorescently modified palmitoylated tripeptides, MTK, was successfully incorporated in the SEVs compositions.
[0167] Therefore, in another embodiment of the present invention, the SEVs compositions are enriched with any of these biologically active molecules according to the method described above and with the respective adaptations for lipid soluble molecules and non-lipid soluble molecules.
4. Compositions Comprising Small Extracellular Vesicles Secreted by UCBMNCs
[0168] Compositions comprising the Small Extracellular Vesicles (SEVs) isolated by the process of the present invention (UF-SEC) were analysed and characterized according to groups of relevant elements contained therein, in particular proteins, RNA and lipids.
1.1 Proteins
[0169] The SEVs compositions were analysed by mass spectrometry to characterize their protein content. Protein concentrations of these compositions were determined by micro bicinchoninic acid assay (BCA) protein assay kit, and the total peptide material analysed using nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Briefly, samples were lysed and loaded onto an (SDS-PAGE gel). The resulting gel lane was washed, and proteins were reduced in Dithiothreitol DTT before alkylation. After washing, in-gel digestion using trypsin was done and peptides were separated by liquid chromatography and identified by MS.
[0170] In total, 170 proteins were identified in the Small Extracellular Vesicles isolated according to the method of the present invention (see Table 1). There is superiority to the new compositions in terms of biological processes associated with regulation of the immune system and inflammatory response (Table 8), since in the prior art method (UC) a low percentage of proteins are associated to these biological processes (Ref. Table 8).
[0171] To understand the biological relevance of these 170 proteins, such as the biological pathways and molecular functions associated, Funrich software was used by introducing their Uniprot IDs and analysing using Funrich database. Table 2 summarizes the percentage of each group in relation to the complete list of proteins identified. Results show that proteins contained in the SEVs compositions have a strong predicted effect in biological processes related to wound healing and immunomodulation.
[0172] The proteins were grouped according to the biological processes that they influence, and Table 3 relates the identified proteins with their biological activity. For example, Funrich software predicted Sp1 as a transcriptional factor influenced by ANXA7, STOM, SLC2A1, DSP, JUP, ANXA11, UBA52 and GAPDH, all of them found in the SEVs composition. This transcriptional factor has been associated with a strong and stable induction of Foxp3 expression when activated which reinforces the possible immunomodulatory effect of the SEVs compositions.
TABLE-US-00001 TABLE 1 List of proteins identified in the SEVs compositions isolated with the present inventive method (UF-SEC) Identified proteins in the SEVs compositions UniProt UniProt UniProt ID Name ID Name ID Name PODOX2 IGA2 P46976 GYG1 P16157 ANK1 PODOX7 IGK P69905 HBA1 P0C0L5 C4B P01871 IGHM P68871 HBB P31944 CASP14 P01619 KV320 P02042 HBD Q16778 HIST2H2BE Q5VTE0 EF1A3 P69891 HBG1 P01859 IGHG2 P01876 IGHA1 P69892 HBG2 Q05707 COL14A1 P04217 A1BG P02008 HBZ Q01518 CAP1 P01023 A2M P62805 HIST4H4 P02549 SPTA1 P63261 ACTG1 P00738 HP P16403 HIST1H1C P12814 ACTN1 P02790 HPX P00736 C1R P43652 AFM Q86YZ3 HRNR PODOY2 IGLC2 P02765 AHSG P11142 HSPA8 P12273 PIP P02768 ALB P08514 ITGA2B Q96P63 SERPINB12 P04075 ALDOA P05106 ITGB3 P07478 PRSS2 P02760 AMBP P01591 JCHAIN P02649 APOE P04083 ANXA1 P14923 JUP Q14254 FLOT2 P50995 ANXA11 P01042 KNG1 Q08188 TGM3 P07355 ANXA2 P11279 LAMP1 P67936 TPM4 P12429 ANXA3 P31025 LCN1 Q06830 PRDX1 P08758 ANXA5 P07195 LDHB 043866 CD5L P08133 ANXA6 Q08380 LGALS3BP P08238 HSP90AB1 P20073 ANXA7 P02750 LRG1 Q9NZD4 AHSP O75882 ATRN P02788 LTF P01768 HV330 P25311 AZGP1 P61626 LYZ Q71DI3 HIST2H3C P30043 BLVRB Q08431 MFGE8 P40926 MDH2 P02745 C1QA P05164 MPO P19338 NCL P02746 C1QB P35579 MYH9 Q93077 HIST1H2AC P02747 C1QC P60660 MYL6 P01031 C5 P09871 C1S Q6UX06 OLFM4 P20618 PSMB1 P01024 C3 P07737 PFN1 O00299 CLIC1 P04003 C4BPA P14618 PKM P09105 HBQ1 P02748 C9 P55058 PLTP Q9UL46 PSME2 P00918 CA2 P02775 PPBP Q86TJ2 TADA2B P04040 CAT P62937 PPIA Q16610 ECM1 Q08722 CD47 P32119 PRDX2 P60900 PSMA6 P08962 CD63 P30041 PRDX6 P53396 ACLY P23528 CFL1 P05109 S100A8 P37802 TAGLN2 P10909 CLU P06702 S100A9 P35754 GLRX P31146 CORO1A P29508 SERPINB3 P29972 AQP1 P00450 CP P05155 SERPING1 P28072 PSMB6 P01040 CSTA P11166 SLC2A1 P28066 PSMA5 P08311 CTSG P02730 SLC4A1 P06331 HV434 P81605 DCD P11277 SPTB O75635 SERPINB7 P59666 DEFA3 P30626 SRI O75368 SH3BGRL Q02413 DSG1 P27105 STOM P59190 RAB15 P15924 DSP P02787 TF Q08554 DSC1 P60842 EIF4A1 P02786 TFRC P01743 HV146 P08246 ELANE P07996 THBS1 P80188 LCN2 P06733 ENO1 Q9Y490 TLN1 P05089 ARG1 P16452 EPB42 P24821 TNC O14818 PSMA7 P21333 FLNA P60174 TPI1 Q6ZVX7 NCCRP1 P02751 FN1 P62987 UBA52 P19652 ORM2 P04406 GAPDH P13611 VCAN P48059 LIMSI P02774 GC P18206 VCL P29353 SHC1 P13224 GP1BB P31946 YWHAB P10599 TXN P06396 GSN P63104 YWHAZ Q5T749 KPRP 015511 ARPC5 P0DOX5 IGG1
TABLE-US-00002 TABLE 2 Biological processes regulated by the proteins contained in Small Extracellular Vesicles isolated according to the method (UF- SEC) of the present invention (percentage of the total proteins identified which are associated to each biological process) % Biological process proteins p-value neutrophil degranulation 20.37 P 0.004 cell adhesion 1.85 P 0.72 extracellular matrix 1.85 P 0.41 organization leukocyte migration 7.41 P 1 cellular protein 3.7 P 1 metabolic process innate immune response 9.26 P 1 regulation of complement 12.96 P < 0.001 activation platelet degranulation 5.56 P 1 Fc-gamma receptor 9.26 P 0.0796 signalling pathway involved in phagocytosis receptor-mediated 9.26 P 1 endocytosis inflammatory response 7.41 P 1 complement activation, 12.96 P 0.002 classical pathway complement activation 11.11 P 0.005 immune response 7.41 P 1 protein deubiquitination 12.96 P 0.187 Fc-epsilon receptor 7.41 P 1 signalling pathway keratinization 5.56 P 1 MAPK cascade 3.7 P 1 NK-kB signalling 10.71 P < 0.001 T cell receptor 10.71 P < 0.001 signalling pathway
TABLE-US-00003 TABLE 3 Biological activity of the identified proteins in the SEVs compositions Biological Number funtion Proteins UniProt ID Code neutrophil 47 P00738; P68871; P05164; P01024; P18206; P07355; P27105; Q02413; P11142; degranulation P04040; P02765; P02788; P05109; P04075; P15924; P30041; P04217; P08311; P14618; Q01518; P02775; Q96P63; P14923; P07478; P02750; P12429; P08238; P08246; P06702; P06396; P61626; Q08722; P29508; P20618; P53396; P28066; O15511; Q08554; P46976; P80188; P05089; P62937; P08962; Q86Y23; P11279; P19652; Q6UX06; platelet 24 P01023; P02768; Q9Y490; P02751; P21333; P02787; P18206; P02765; Q08380; degranulation P04075; P10909; P08514; P04217; P12814; P02775; P07996; P08758; P05106; P01042; P05155 Q16610; P37802; P08962; P19652; complement 19 P01871; P01024; P02748; P02747; P0C0L5; P02746; P04003; P10309; P01876; activation, P01859; P02745; P09871; P00736; P01768; P01031; P05155; P01619; P06331; classical pathway P01743; innate immune 19 P01871; P04063; P02747; P0C0L5; P05109; P62987; P02746; P04003; P10909; response P01876; P59666; P01859; P02745; P00736; P01591; P06702; P05155; P31146; P80188; cell adhesion 15 P02751; P24821; P18206; P13611; Q08380; Q06431; P25311; P08514; P07996; Q14254; P05106; Q08722; P13224; P02760; Q6UX06; complement 14 P01024; P02747; P0C0L5; P02746; P10909; P01859; P02745; P09871; P00736; activation P01768; P01031; P01619; P06331; P01743; receptor-mediated 14 P02768; P00738; P69905; P68871; P02790; P01876; Q01518; P02649; P01591; endocytosis P01768; P01619; P06331; P01743; P02790; P02760; inflammatory 13 P01024; P04083; P0C0L5; P05109; O75882; P02775; P07996; Q43866; P06702; response P61626; P01042; P01031; Q16610; leukocyte 13 P02751; P01871; P35579; P01876; P01591; P05106; P01768; Q08722; P01619; migration P06331; P01743; P62937; P29353; negative 13 P02768; P05164; P21333; P32119; P63104; P04083; P04040; P02788; P62987; regulation of P07996; P08758; P29972; P23528; apoptotic process Fc-epsilon 12 P62987; P01768; P20818; P01619; Q9UL46; P60900; P28072; P28066; P06331; receptor P01743; Q14818; P29353; signaling pathway immune response 12 P01024; P02747; P01876; P08311; P02775; P00736; P07996; P01591; P01768; P01619; P06331; P01743;
[0173] To refine the proteomic analysis, SEVs samples were segmented in three fractions increasing the dynamic range of MS analysis to identify the less abundant peptides. In addition to the above listed proteins, SEVs purified by the process of the present invention (UF-SEC) are also composed by classical SEVs markers as tetraspanin like CD9, CD63 and CD82, integrins (integrin alpha-2; integrin alpha-IIb; integrin alpha-4; integrin alpha-M; integrin beta-1; integrin beta-2 and integrin beta-3) and Rab proteins.
2.2 RNA
[0174] RNA sequencing was used to identify the small RNA content of the SEVs compositions. SEVs RNA was isolated using EXIQON miRCURY™ Isolation kit (Cell & Plants) and small RNA quality and quantification was performed in Bioanalyzer 2100.
[0175] In the scope of the present invention the expression “small RNA content” means a composition comprising RNA molecules having less than 200 nucleotides in length, and “miRNA”, “microRNA” or “miR” means a RNA molecule with approximately 21-25 nucleotides.
[0176] A small library was constructed using small RNA according to Ion Total RNA-Seq Kit v2 protocol. The pooled libraries were further processed on Ion Chef™ System and the resulting 530™ chip was sequenced on Ion S5™ System. Cufflinks tool was used for annotation and estimation of the relative abundance of each gene in each sample.
[0177] Results show that SEVs isolated according to the process of the present invention have different species of small RNAs, with transference RNA being the most abundant RNA species, representing in average 57%. MicroRNAs (miRNA) are the second most predominant specie (˜27%) followed by ribosomal RNA (˜6%) and other minor categories. Table 4 shows the small RNAs identified in the SEVs compositions and their correspondent contribution in respect to the total of annotated RNA sequences.
TABLE-US-00004 TABLE 4 Percentage of small RNA species in the SEVs compositions AVERAGE Standard SMALL RNA (% of total annotated) Deviation snRNA 1.99 1.08 snoRNA 1.47 0.98 rRNA 6.19 0.97 microRNA 26.47 5.40 tRNA 57.23 6.87 Other 6.65 1.23
[0178] Amongst the 6 analysed samples, there are no major differences in RNA species. Accordingly, a range of miRNA can be defined as shown in Table 5. In the SEVs compositions, 227 RNAs were detected in at least 5 different samples, of which 44 are miRNA. Interestingly, 20 of those miRNA identified are exclusive of SEVs isolated with the new inventive method (miRNAs in bold in table 5).
TABLE-US-00005 TABLE 5 List of the miRNA commonly identified in the SEVs compositions. In bold, in the bottom of the list are presented miRNA species identified exclusively in SEVs preparations isolated with the inventive method. miRNA identified hsa-let-7a-5p hsa-miR-15a-5p hsa-miR-21-5p hsa-let-7f-5p hsa-miR-15b-5p hsa-miR-223-3p hsa-let-7g-5p hsa-miR-16-5p hsa-miR-23a-3p hsa-miR-103a-3p hsa-miR-17-5p hsa-miR-26a-5p hsa-miR-106b-5p hsa-miR-191-5p hsa-miR-26b-5p hsa-miR-142-3p hsa-miR-19a-3p hsa-miR-29b-3p hsa-miR-146a-5p hsa-miR-19b-3p hsa-miR-30d-5p hsa-miR-150-5p hsa-miR-20a-5p hsa-miR-451a hsa-let-7b-5p hsa-miR-185-5p hsa-miR-30b-5p hsa-let-7c-5p hsa-miR-18a-5p hsa-miR-3184-3p hsa-let-7i-5p hsa-miR-205-5p hsa-miR-376c-3p hsa-miR-130a-3p hsa-miR-221-3p hsa-miR-486-5p hsa-miR-144-3p hsa-miR-22-3p hsa-miR-92a-3p hsa-miR-144-5p hsa-miR-27a-3p hsa-miR-93-5p hsa-miR-181a-5p hsa-miR-27b-3p
1.3 Lipids
[0179] Given that Small Extracellular Vesicles are surrounded by a lipid bilayer, they are considered not only an important structural component of Small Extracellular Vesicles, but they are also expected to modulate Small Extracellular Vesicles interaction with recipient cells. To have some insight regarding the lipid content of the compositions of the present invention mass spectrometry was used to measure and identify their lipid profile. Lipids were extracted using chloroform and methanol, according to the methodology of Sampaio J L, PNAS 2011.
[0180] Samples were spiked with lipid class-specific internal standards prior to extraction. After drying and resuspending in Mass Spectrometry acquisition mixture, lipid extracts were subjected to mass spectrometric analysis. Mass spectra were acquired on a hybrid quadrupole/Orbitrap mass spectrometer equipped with an automated nano-flow electrospray ion source in both positive and negative ion mode.
[0181] In terms of total lipid quantity, it was observed that the compositions have approximately 12000 pmol with a variability between samples of 25%, with phosphatidylcholines (PC) and phosphatidylserines (PS) as the major lipid classes (˜60%), followed by Sphingomyelin (SM, 20%) as shown in Table 6.
TABLE-US-00006 TABLE 6 Percentage of lipids in SEVs compositions. CE- cholesteryl esters; DAG-diacylglycerol; PA-phosphatidic acid; PC-phosphatidylcholine; PE-phosphatidylethanolamine; PG-phosphatidylglycerol; PI-phosphatidylinositol; PS-phosphatidylserine; SM-sphingomyelin; TAG- triacylglycerol. Percentage of Lipid Total Lipids (%) CE 0.05 ± 0.05 DAG 1.3 ± 0.4 PA 3.18 ± 0.6 PC 30.9 ± 3.3 PE 13.0 ± 0.5 PG 0.25 ± 0.1 PI 3.8 ± 0.3 PS 29.4 ± 3.7 SM 18.0 ± 1.6 TAG 0.074 ± 0.13 Total 100
[0182] The levels of Triacylglycerides (TAG) and Diacylglycerides (DAG) are low, which indicate that the SEVs compositions isolated with the new inventive process are essentially not contaminated with lipoproteins.
[0183] Considering that these compositions contain high amounts of PC and SM (˜50%), which both include choline groups, quantification of cholines was also included in the characterization of SEVs. For this purpose, a commercial kit with high sensibility for cholines can be used. In the present case the Phospholipid Assay Kit® MAK122-1KT from Sigma-Aldrich was used.
[0184] It was observed that the average concentration of cholines in the SEVs compositions is 25 uM, which corresponds approximately to 15 uM of PC and 10 uM of SM, considering their corresponding percentage as evaluated by Mass Spectrometry (Table 6). These results show that SEVs isolated with the inventive method have a lipid composition in accordance to what is described in the literature for SEVs isolated by prior art method.
[0185] In consequence, it is acknowledged that this quantification methodology may function as a measure of the quality of the compositions in function of their lipid content.
[0186] Therefore, in another embodiment the present invention refers to the use of a high sensibility for cholines, such as the above-mentioned Phospholipid Assay Kit® MAK122-1KT or similar for measurement of the quality of the compositions in function of their lipid content.
[0187] Another embodiment of the present invention relates to compositions comprising the SEVs isolated by the process of the present invention, in particular SEVs compositions comprising these three groups of molecules with biological activity.
[0188] In a preferred embodiment, the SEVs compositions comprise the 170 proteins listed in Table 1, in particular ANXA2, ANK1, CD63, CD81, CD9 and CD15.
[0189] In another preferred embodiment, the proteins in the SEVs compositions consist essentially in one or more selected from the group the 170 proteins listed in Table 1, in particular in at least one of the proteins selected from the group ANXA2, ANK1, CD63, CD81, CD9 and CD15.
[0190] In a more preferred embodiment proteins at least one of the following proteins are present in SEVs compositions in an amount of: CD81≥1%, CD9≥1%, CD63≥40%, CD15≥20% positive events as measured by flow cytometry of SEVs coupled to microbeads, or quantity of CD63≥5 pg/mL or ANXA2≥0.3 ng/mL in the purified SEVs, as measured by ELISA.
[0191] In another embodiment the SEVs compositions comprise micro RNA present in Table 4, preferably miR-150-5p, miR-223-3p, miR-16-5p, miR-142-3p, miR-19b.
[0192] In another preferred embodiment, the micro RNA present in the SEVs compositions consist in at least of one micro RNA as presented in Table 4, more preferably, consist in one or more micro RNA selected from the group of miR-150-5p, miR-223-3p, miR-16-5p, miR-142-3p, miR-19b.
[0193] In another preferred embodiment, the SEVs compositions comprise at least one of the following micro RNAs in the respective amounts: miR-150-5p>1.3 pg/10{circumflex over ( )}9 part, miR-223-3p>1.2 pg/10{circumflex over ( )}9 part, miR-16-5p>0.5 pg/10{circumflex over ( )}9 part, miR-142-3p>0.4 pg/10{circumflex over ( )}9 part, miR-19b>0.2 pg/10{circumflex over ( )}9 part, as measured by direct quantification.
[0194] In another preferred embodiment, the micro RNA present in the SEVs compositions consist in at least of one micro RNA selected from the group of the following and respective amounts: miR-150-5p>1.3 pg/10{circumflex over ( )}9 part, miR-223-3p>1.2 pg/10{circumflex over ( )}9 part, miR-16-5p>0.5 pg/10{circumflex over ( )}9 part, miR-142-3p>0.4 pg/10{circumflex over ( )}9 part, miR-19b>0.2 pg/10{circumflex over ( )}9 part, as measured by direct quantification.
[0195] In another embodiment, the lipids in the SEVs compositions consist essentially in one or more selected from the group the lipids listed in Table 6, i.e. CE, DAG, PA, PC, PE, PG, PI, PS, SM, TAG.
[0196] In another embodiment, said lipids are present in the SEVs compositions in a percentage, respective to the total lipid concentration, as listed in Table 6, i.e. CE≥0.05_±0.05, DAG≥1.3±0.4, PA≥3.18±0.6, PC≥30.9±3.3, PE≥13.0±0.5, PG≥0.25±0.1, PI≥3.8±0.3, PS≥29.4±3.7, SM≥18.0±1.6, TAG≥0.074±0.13.
[0197] In a preferred embodiment, the SEVs compositions comprise or consist respective to the 3 groups of molecules mentioned above and with biological activity, in: [0198] Proteins identified and respective amounts of Table 1 and 3; [0199] RNAs identified and respective amounts of Table 4 and 5, and [0200] Lipids of Table 6.
[0201] In another preferred embodiment, the SEVs compositions comprise or consist respective to the 3 groups of molecules mentioned above and with biological activity, in a combination of at least one of the following: [0202] Proteins: ANXA2, ANK1, CD63, CD81, CD9 and CD15; [0203] RNAs: preferably miR-150-5p, miR-223-3p, miR-16-5p, miR-142-3p, miR-19b, and [0204] Lipids of Table 6, i.e. CE, DAG, PA, PC, PE, PG, PI, PS, SM, TAG.
5. Biological Effect of the Compositions
5.1 In Vitro Anti-Inflammatory Impact on Macrophages
[0205] Monocytes and macrophages possess broad immuno-modulatory, inflammatory, and tissue-repairing capabilities and actively participate in the development of many autoimmune and diseases. In fact, infiltration and exaggerated activation of these cells is generally observed in many autoimmune diseases as well as inflammatory diseases such as type 1 diabetes, rheumatoid arthritis and others.
[0206] SEVs biological effect on inflammation was evaluated in an in vitro model composed by macrophages differentiated from a monocytic cell line (THP-1) by the action of a chemical compound, phorbol 12-myristate-13-acetate (PMA).
[0207] Differentiated macrophages were incubated with 1×10.sup.10 part/mL of SEVs and after 24 hours, their phenotype was evaluated as well as the mRNA levels of three major cytokines involved in pro-inflammatory process were evaluated (
[0208] In response to different stimuli, macrophages can acquire two different major phenotypes: M1 (pro-inflammatory) and M2 (anti-inflammatory). The classically activated or M1 macrophage phenotype produces inflammatory cytokines such as TNF-α, which trigger acute inflammation, while the alternatively activated M2 phenotype is anti-inflammatory and has been found to play an important role in tissue and wound repair.
[0209] In the present invention, CD86 and CD163 staining was used to distinguish between M1 and M2 phenotype, respectively according to the described by Martinez and Gordon (2014). It was observed a decrease in M1/M2 ratio in macrophages (CD14.sup.+) treated with the SEVs isolated by the process of the invention indicating a higher prevalence of the anti-inflammatory macrophages (
[0210] In comparison with the vehicle (PBS), SEVs decreased the mRNA expression of IFN-γ, IL1β, TNF-a and COX-2 (
[0211] We then decided to evaluate if SEVs could counteract the progression to a chronic inflammatory state. For that we used THP-1 derived macrophages stimulated with LPS, and co-incubated with SEVs (
[0212] In this model, SEVs were able to decrease M1/M2 ratio with a higher impact than in the previous one (
[0213] Additionally, we observed a significant decrease of TNF-α secretion in THP-1 derived macrophages, indicating that this cell model become less inflammatory.
[0214] These results indicate that SEVs may induce resolution of the inflammatory phase. Resolution of inflammatory phase would benefit not only skin inflammatory diseases as well as other systemic disease whose pathogenicity is centred on chronic inflammation.
5.2 In Vitro Immunomodulatory Effect of SEVs on Peripheral Blood Mononuclear Cells (PBNNCs)
[0215] With the aim to assess the immunomodulatory potential of the compositions of the present invention, samples of healthy human peripheral blood mononuclear cells (PBMNCs) with SEVs compositions of the present invention were analysed and T cell proliferation and polarization was evaluated.
[0216] A composition comprising SEVs and obtained according to the method of the present invention, as described in section 1, was applied to 3 donor samples of PBMNCs at 1×10.sup.10 part/mL concentration. 10.sup.6 PBMNCs per well were cultured in 24 well plate with depleted-RPMI 1640 complete medium and after 6 days of incubation, T cell proliferation and the prevalence of Treg (positive for CD25 and FoxP3) was evaluated within T cell subpopulations by flow cytometry. Cells were washed, fixed and permeabilized before antibody staining.
[0217] A decrease in overall T lymphocyte population (CD3+ cells)—58.7±3.8% versus 38.9±6.6%—as well as in CD4.sup.+ T lymphocyte subset—42.9±2.1% versus 28.9±6.5%—and CD8+ T cells—66.2±4.1% versus 41.54±14.0% was observed upon treatment with SEVs composition (
[0218] In addition, SEVs compositions were able to significantly increase the percentage of Treg in cultured samples (9.68±1.89% in treated, against 5.24±2.53% in control—culture without SEVs compositions (
[0219] To reinforce the idea of a rebalance in Treg/Teff ratio, IL-7 receptor a (CD127) was used as a negative marker of Treg since its expression is inversely correlated with FoxP3 activity and it is reported in the majority of effector and memory T cells. In this experiment, SEVs were able to increase Treg population within CD4.sup.+ cells while decrease the percentage of effector T cells (
[0220] There is a direct correlation between IFN-γ secretion and the level of T cell activation. In order to determine the effect of SEVs on the secretory IFN-γ response of T cells, PBMNCs were cultured in the presence and absence of SEVs for 6 days and intracellular levels of IFN-γ were determined on CD4.sup.+ and CD8′ T cell subsets. Results show that, at day 6, the percentage of intracellular IFN-γ was reduced when PBMNCs were cultured with SEVs, in comparison to positive control, in both T cell subsets. However, this reduction was only statistically significant on gated CD4.sup.+ T cells (
[0221] Additionally, T helper cell polarization was evaluated by quantifying the expression of lineage genes in the lymphocyte subset at 2 and 6 days after SEVs administration. From the PBMNCs mixture, lymphocytes were sorted based on their size and complexity and total RNA was extracted, converted to complementary DNA and final analysed by quantitative PCR (qPCR). For this purpose, master transcription factors were evaluated for each lineage: GATA3 for Th2; T-bet for Th1; Foxp3 for Treg; and RORYT for Th17, according to the method described in Zhu, Yamane, and Paul (2010).
[0222] PBMNCs were incubated with SEVs compositions at 1×10.sup.10 part/mL, and at day 6 (day zero is the day of administration), the expression of several transcription factors was quantified (
[0223] It was observed that the SEVs induce a differentiation to the Treg phenotype, with increased expression of FoxP3 (
[0224] Surprisingly, it was observed a significant inhibition of RORYT, which suggest decreased expansion of Th17 cells upon treatment with SEVs compositions (
[0225] Finally, the SEVs potential was evaluated on the secretion of cytokines. Without any stimulus, we were not able to detect the secretion of any small molecule. However, lipopolysaccharide (LPS) stimulation highly increased the secretion. This way when stimulated simultaneously with LPS and SEVs, PBMNCs mixture significantly decrease TNF-α secretion. TNF-α is a pro-inflammatory cytokine that is involved in the regulation of immunity and several inflammatory diseases in humans. As it plays a critical role in the pathogenesis of autoimmune inflammatory diseases, current biological therapies are designed to block its biological function. Added to SEVs potential of increase Treg in the system, the decrease TNF-α secretion is another indication of SEVs immunomodulatory effect as well as a future autoimmune therapy.
[0226] Additionally, CCL20 secretion was also tested. One of the main effects of CCL20 is to promote the recruitment of CCR6.sup.+ lymphocytes. CCR6 is expressed by a variety of cells of the innate and/or adaptive immune system, in particular T-helper 17 (Th17) cells, which exert multiple functions via release of cytokine IL-17A in many chronic inflammatory disorders. Again, the decrease of in vitro production of this chemokine contributes to the immunomodulatory capacities of SEVs.
[0227] Evidence was shown that the SEVs compositions of the invention present immunotolerant properties, namely through T cell differentiation towards a regulatory phenotype.
[0228] To further understand the mechanism of action of these compositions, two different microRNAs (miR) were directly transfected into a PBMNCs mixture. miR-146-5p is highly expressed in regulatory T cells. It is indispensable for their immunosuppressive function, since its deletion results in a breakdown of immunological tolerance manifested in an IFNy-dependent immune-mediated lesions in a variety of organs. miR-150-5p is reported as Treg differentiation driver in conjugation of other miRs although its role is demonstrated through induction and proliferation of CD8.sup.+ effector cells.
[0229] Regarding miR-146a-5p, results showed a significant increase on T-bet and FoxP3 expression (
[0230] Surprisingly, miR-150-5p transfection resulted in an overexpression of RORYT and FoxP3, which shows that this miR induces the differentiation into a new regulatory T cell subtype that was recently described as T regulatory 17 FoxP3.sup.+RORYt.sup.+, which is able to suppress Th17 differentiation and control autoimmunity.
[0231] In parallel with gene expression analysis, the percentage of Treg cells was quantified by flow cytometry, and it was observed an increase in regulatory T cell subset in PBMNCs transfected with both miR (
[0232] In Short, it was Demonstrated that SEVs Compositions: [0233] reduce/attenuate T cell proliferation; [0234] induce the differentiation of regulatory T cells; [0235] differentially influence mRNA expression in lymphocytes; [0236] differentially influence FoxP3 mRNA expression in PBMNCs; [0237] decrease the secretion of pro-inflammatory cytokines; [0238] miR-150-5p and miR-146-5p show substantial impact on lymphocytes expression profile; [0239] miR-150-5p and miR-146-5p show substantial impact on lymphocytes phenotype
[0240] These results show that SEVs compositions of the present invention have an advantage effect on immune cells and therefore, said compositions are useful to be applied to treat or prevent diseases related with imbalance or overreaction of the immune system.
5.3 Effect in Psoriasis—In Vitro
[0241] Based on above described previous results that demonstrated that SEVs compositions are able to not only increase the number of Treg but also decrease the expression of RORYT, a master transcriptional factor of Th17 T cell lineage, the impact of compositions in models of psoriatic epidermis were studied.
[0242] For that purpose, first keratinocytes were differentiated with Ca.sup.2+ to mimic the epidermis outermost layer and applied 5% of Imiquimod (IMQ), a compound described to resemble most of the psoriasis phenotype, through its proinflammatory properties.
[0243] After 3h of simultaneous incubation (SEVs compositions and IMQ), a decreased expression of TNF-cx and an increased expression of VEGF-a and TGF-b1 was observed in response to the application of SEVs compositions (
[0244] TNF-α is a pro-inflammatory cytokine that amplifies inflammation through several distinct pathways: facilitating entry of inflammatory cells into lesioned skin through induction of adhesion molecules on vascular endothelial cells; stimulating keratinocyte production of other pro-inflammatory mediators and activating dermal macrophages and dendritic cells. Thus, its inhibition promoted by the SEVs compositions can ameliorate psoriasis pathophysiology by blocking the vicious activating cycle of dendritic cells.
[0245] On the other hand, these compositions stimulated VEGFA and TGFb1 expression. VEGFA plays a key role in the establishment of an immunosuppressive tumour microenvironment, as it inhibits the maturation of dendritic cells, and stimulates the proliferation of Tregs.
[0246] Also, TGF-b1 has been shown to limit inflammation and to control peripheral tolerance by both direct and indirect regulation of autoreactive T cells. Although TGF-β is essential for T cell tolerance, it has recently been proposed that loss of TGF-β signalling alone is insufficient to induce autoimmunity.
[0247] In a similar model, SEVs compositions stimuli potentiated the downregulation of IL-6 expression when compared to the vehicle (
Results Show that SEVs Compositions: [0248] decreases expression of pro-inflammatory molecules such as TNF-a and IL-6 in psoriatic like-NHEK model; [0249] potentiate VEGFA and TGFb1 expression in psoriatic like-NHEK model.
[0250] Therefore, it was also demonstrated that SEVs compositions of the present invention are useful to be applied to psoriasis treatment or prevention.
5.4 Effect in Psoriasis-Like Epidermis
[0251] To confirm the potential anti-psoriasis effect in skin, a commercial three-dimensional epidermis model (PSORIASIS LIKE EPIDERMIS, Sterlab) was used. The model was treated for 5 days with a single dose of SEVs a composition every day. At the end of experiment, cells were collected for RNA analysis and supernatant for protein secretion quantification.
[0252] Results show that the SEVs composition downregulates the mRNA expression of several cytokines such as IL-6, IL-8, TNF-a and IFN-g (
[0253] Additionally, the expression of antimicrobial peptides (S100A7 and DEFB4) was analysed and found consistent downregulation of both (
[0254] Furthermore, these antimicrobial peptides contribute to chemotaxis of neutrophils and T cells, exacerbating psoriasis phenotype. Decreasing the expression of these peptides significantly contributes to ameliorate the psoriatic pathophysiology.
[0255] Moreover, SEVs compositions do not seem to impact the differentiation stage of keratinocytes as no differences were seen on KRT5 (
[0256] When analysing protein secretion, it was observed that the application of SEVs compositions decreases CCL20 and TNF-α (
[0257] Results show that SEVs described in the present invention promote a robust anti-psoriasis effect, by influencing several molecular pathways, which sustain psoriasis and other auto-immune diseases. The results suggest that SEVs may be used as an anti-inflammatory for human diseases with epidermis involvement.
5.5 Effect in Psoriasis—In Vivo
[0258] To access the efficacy of the SEVs compositions on a psoriatic in vivo model, IMQ-induced skin inflammation was chosen as a model that phenotypically resembles psoriasis in mice, according to the method described in van der Fits et al. (2009).
[0259] The model was induced by topical application of Aldara@, a cream preparation containing 5% IMQ, every day for 5 days. IMQ is a ligand for the toll-like receptors TLR7 and TLR8 that exacerbates psoriasis at both the local treated areas as well as distant sites. TLR7 and TLR8 are expressed by monocytes, macrophages and dendritic cells that when are activated lead to pro-inflammatory cytokines and chemokines production. Application of Aldara® on hair-free back of mice results in the development of psoriasis-like lesions within 5 days of application and is underpinned by an influx of various cells as well as hyperplasia of the epidermis.
[0260] During the six days of consecutive topical treatment with SEVs compositions, the mice skin clinical score was monitored. The final value resulted in a sum of three parameters: severity of erythema (redness from 0 to 4), affected area (0-3) and desquamation (scale from 0-2). Topic application of SEVs compositions of the inventions delayed the appearing of psoriatic characteristics and attenuated them over time (
[0261] Additionally, topical treatment with said compositions significantly decrease the number of skin infiltrating neutrophils (CD45.sup.+CD11b.sup.+Ly6G.sup.+CD64.sup.−) and mature T lymphocytes (CD45.sup.+CD3.sup.+TCRαβ.sup.+) (
[0262] Activated T cells produce abundant cytokines like IL-17, IFN-y, TNF, and IL-22 while neutrophils secrete chemokines, such as CXCL1, CXCL2, and CXCL8/IL-8. Both cytokines and chemokines contribute to psoriatic vicious cycle by polarizing de novo Th1 and Th17 immune cells and activating keratinocyte hyperproliferation. There is tendency for a decrease on the presence of inflammatory macrophages (
[0263] Moreover, it is generally accepted that the pathogenesis of psoriasis involves accumulation of effector T-cells within lymph nodes and their subsequent migration into the skin through the blood system. Results show that Th17 cells and Gamma-delta expressing cells were significantly decrease in the mice lymph nodes treated with SEVs compositions (
[0264] Besides the decrease of Th17 cells, mRNA RORgt levels were also decrease in lymph node samples of treated animals with SEVs (
[0265] Additionally, and because psoriasis is a CD8 driven disease, the decrease of abundance of these cells both at skin (
[0266] Therefore, the SEVs compositions of the present invention are able to ameliorate psoriasis-like skin inflammation in an in-vivo model.
6. Effect in Tissue Repair—Wound Healing In Vivo
[0267] We decided to validate if the inventive process of isolation of SEVs would have an impact in their bioactivity and potential to treat skin wounds.
[0268] The results show that the SEVs isolated with the prior art method (UC—ultracentrifugation) are less bioactive than the ones isolated with inventive method UF-SEC (
[0269] In conclusion, the present invention relates to a process for isolating Small Extracellular Vesicles secreted by umbilical cord blood cells (UCBCs), including isolating Small Extracellular Vesicles from umbilical cord blood mononuclear cells (UCBMNCs), and compositions comprising Small Extracellular Vesicles obtained by processing said UCB fraction, which is useful to be applied to inflammatory or autoimmune diseases therapeutics or prophylactics and/or cosmetic purposes.
[0270] Accordingly, the present invention also relates to Small Extracellular Vesicles pharmaceutical compositions useful to be applied in therapy, in cosmetics and/or in prophylactic methods.
[0271] Therefore, in one embodiment of the invention the SEVs as described above are used to produce a pharmaceutical composition to be applied as cosmetics, as a medicament and/or as prophylactic agent.
[0272] In another embodiment, the SEVs as described above are used to produce a pharmaceutical composition to be applied in autoimmune-disease therapy including in skin autoimmune-disease therapy.
[0273] In another embodiment, the SEVs as described above are used to produce a pharmaceutical composition to be applied as medicament in tissue repair including skin tissue repair, skin open wound therapy, such as an incision, a laceration, a tear, an abrasion, an avulsion or a surgical wound, skin burn therapy, such as a thermal burn, a chemical burn or a radiation burn, and/or a skin condition or disease, such as acne, psoriasis, rosacea, dermatitis, eczema, impetigo, intertrigo, or folliculitis, in wound therapy such as arterial ulcers, venous ulcers, diabetic ulcers and pressure ulcers, post-surgical ulcer, traumatic ulcer, mouth ulcer, diabetic foot ulcer or corneal ulcer, etc.
[0274] In another embodiment, the SEVs as described above are used to produce a composition in a liquid form, in a spray form, in a cream form, in a gel form, being the liquid form as an edible solution, external applicable solution, internal applicable solution or injectable solution.
[0275] In another embodiment, the SEVs as described above are used to produce a composition supported by a carrier such as a natural or synthetic polymer matrix, a patch or another suitable medical carrier.
EXAMPLES
Example 1. PBNKCs Collection and Processing
[0276] Human blood samples were obtained from the Hospital Universitário de Coimbra, where donations were collected from healthy volunteers after providing their informed consent. PBMNCs were isolated by Lymphoprep™ gradient centrifugation. PBMNCs were frozen and stored in liquid nitrogen. For in vitro experiments, cell aliquots were thawed at 37° C., added to 10 mL of RPMI 1640 and centrifuged at 300 g for 5 min to eliminate DMSO. Pellet was resuspended in RPMI 1640 supplemented with 10% of FBS. PBMCs were plated on 96-well flat bottom culture plates (Corning-Costar, Celbio, Milan, Italy) at the density of 2-3×10.sup.5 cells/well and cultured with RPMI 1640 medium containing 10% FBS.
Example 2. Gene Expression Analysis
[0277] RNA samples were extracted by RNAeasy Mini kit (Qiagen) according to manufacture recommendation. RNA concentration was quantified with total RNA chips in Bioanalyzer 2100 (Agilent). All RNA samples were stored at −80° C. RNA was reverse-transcribed according to manufacturer's instructions of SuperScript IV VILO Master Mix (Invitrogen). After reverse transcription, the cDNA was immediately used for qPCR or were preserved at −20° C. qPCR was performed using the NZYSpeedy qPCR Green Master Mix (2×), ROX in a 96-well format in CFX96 Touch™ Real-Time PCR Detection System (BioRad).
[0278] The following conditions, 95° C. for 2 min, followed by 40 cycles at 95° C. for 5 s, 60° C. for 30 s, 72° C. for 20 s (measuring the fluorescence) were used for the qPCR. At least three biological replicates were used. β-actin (ACTB) was used as an endogenous control to normalize each sample. The resulting data was analysed using Bio-Rad CFX Manager software. The relative expression of interest genes was analysed according to the 2.sup.−ΔΔCt method.
Example 3. Flow Cytometry
[0279] PBMNCs were collected and erythrocytes were osmotically lysed in red blood cell lysis buffer. For cell surface staining, single-cell suspensions were incubated for 30 min with saturating concentrations of mAbs. Cells were stained for the above identified cell surface markers, fixed 30 min at 4° C., permeabilized with the Foxp3/Transcription Factor Staining Buffer set (eBioscience) in the presence of anti-CD16/CD32 (eBioscience) for 15 min at 4° C., and lastly incubated for 1 hour at 4° C. with the above identified antibodies in permeabilization buffer. Samples were acquired using BD accuri C6 (BD Biosciences). Data were analysed using FlowJo 7.0.0. software.
Example 4. UCBMNCs Culture
[0280] PBMNCs were collected and erythrocytes were osmotically lysed in red blood cell lysis buffer. For cell surface staining, single-cell suspensions were incubated for 30 min with saturating concentrations of mAbs (Table 2). Cells were stained for the above identified cell surface markers, fixed 30 min at 4° C., permeabilized with the Foxp3/Transcription Factor Staining Buffer set (eBioscience) for 15 min at 4° C., and lastly incubated for 1 hour at 4° C. with the above identified antibodies in permeabilization buffer. Samples were acquired using BD accuri C6 (BD Biosciences). Data were analyzed using FlowJo 7.0.0. software.
Example 5. PBNNCs Transfection
[0281] In order to transfect PBMNCs with miRNAs Viromer® transfection reagents (Lipocalyx, Germany) were used. Viromer® Blue and Viromer® Green are polymer-based transfection reagents featuring a viral mechanism of membrane fusion. They do form transfection complexes with small interfering RNA (siRNA) or microRNA (miRNA) having a size of a few hundred nanometers. Viromer®:RNA complexes are taken up by endocytosis, a process that involves the formation of an acidic compartment. The low pH in late endosomes acts as a chemical switch that renders the Viromer® surface hydrophobic and facilitates membrane crossing. This “Active Endosome Escape” technology is safe and maximizes transfection efficiency as it is using a natural uptake pathway. Once reached the cytoplasm, Viromer complexes disaggregate—releasing RNA or DNA. The innovative Viromer® complexes are non-charged, preventing the aggregation with proteins which are included in foetal calf serum or other cell culture media supplements. This leads to the great advantage, that Viromer® is very gentle on cells, compatible with serum, antibiotics and differentiation factors.
[0282] Viromer® Blue and Viromer® Green are optimized for the in vitro transfection of siRNA and miRNA. They are highly effective on a wide range of standard and hard-to-transfect cells including suspension cells, stem cells and primary cells.
[0283] The transfections were performed accordingly to manufacturer's recommendations. Briefly, we pre-plated PBMNCs in 12-well plates at a concentration of 1×10.sup.6 cells/ml (RPMI medium, no serum, no antibiotics). We then prepared a dilution of 10 μM miR-mimic in Viromer buffer and mixed it with Viromer transfection reagent. After 15 min at room temperature, the transfection complex was added to cells that were further incubated up to 6 days (miR-mimic final concentration was 100 nM).
Example 6. Statistical Analysis
[0284] Bar graphs are showed as mean±standard error of mean (SEM). The statistical significance was assessed by two-tailed unpaired Student's t test, one-way ANOVA with Tukey's multiple comparison or two-way ANOVA with Bonferroni multiple comparison post-test, as required. All analysis was performed with GrapPhad software. Differences were considered significant at p<0.05.
Example 7. Isolation of Small Extracellular Vesicles (SEVs)
[0285] UCB units were automatically processed by using an automated system AXP, according manufactures' recommendations. Briefly, cord blood is transferred into the processing bagset which is placed in the AXP device and then centrifuged. The AXP device fits into most standard blood bank centrifuge buckets allowing up to six samples to be processed simultaneously. During centrifugation, component stratification and separation occurs. RBCs are transferred to a separate sterile bag, the buffy coat which contains the MNC rich layer is delivered to a separate sterile freezing bag while the plasma remains in the processing bag (http://cescatherapeutics.com/blood-marrow-processing/axp-autoxpress/).
[0286] The MNCs obtained from the Umbilical Cord Blood (2.000.000 cells/mL) were cultured in X-VIVO 15 serum-free cell-culture medium (Lonza Group Ltd, Basel, Switzerland) supplemented with 0.5 ug/mL of FMS-like tyrosine kinase-3 and 0.5 ug/mL of stem-cell factor, under ischemia (0.5% O2) conditions.
[0287] After 18 hours of secretion the conditioned media was carefully removed to a falcon to proceed with the SEVs purification.
[0288] The conditioned media was cleared by 2 sequential centrifugation steps of 300 g (10 minutes) and 2000 g (20 minutes). After these steps the supernatant was collected and subject to two filtration steps using a syringe, first using a 0.45 uM filter and afterwards a 0.22 um. After this initial clearance the media was subject to a final ultrafiltration step using a pressure-controlled device (VivaCell 250).
[0289] The supernatant was subject to a pressure of 3 bar through a 100 KDa filter in a Vivacell (Sartorius) device and the supernatant containing the SEVs was recovered in the top compartment after a washing step with filtered PBS. Then, 13-15 mL of the concentrated solution were subject to a size exclusion chromatography using a Superose 6 pre-packed XK 26/70 (GE Healthcare), respectively in a FPLC− Akta Avant Chromatographic system. A Filtered PBS solution was used as elution buffer at a flow rate of 3 mL/min (XK 26/70) and UV absorbance was detected at three wavelengths: 220, 260 and 280 nm. Fractions of 5 mL (XK 26/70) were collected, using an automatic fraction collector coupled with the equipment, for analysis by NTA and Micro BCA.
Example 8. Small Extracellular Vesicles (SEVs) Characterization
8.1. Protein Characterization
[0290] SEVs isolated by the process of the present invention, referred herein as UF or INV process, were analysed by mass spectrometry to characterize their protein content. Protein concentrations were determined by micro BCA protein assay kit (Thermo Scientifics TM) and approximately 0.5 μg total peptide material was analysed using nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Briefly, samples were lysed and loaded onto an SDS-PAGE gel. The resulting gel lane was washed, and proteins were reduced in DTT before alkylation. After washing, in-gel digestion using trypsin was done and peptides were separated by liquid chromatography and identified by MS. These proteins are listed in Table 1 and Table 2 in the Description Section.
[0291] From the three analysed pools of SEVs isolated by UF-SEC (each pool obtained from 5 UCB, totalizing 15 UCB), 31 proteins were identified as common between pools, representing in average 61% of the total identified protein content. Within this group, albumin and different haemoglobin subunits have the highest number of identified peptides, followed by band 3 anion transport protein and haptoglobin.
[0292] These proteins are essential constituents of human umbilical cord blood and their function is associated with oxygen delivery by red blood cells. Although MS is a very powerful tool and detects a high number of proteins in one single run, its sensitivity is defined by the dynamic range of the samples and proteins with a low expression may not be detected. We believe that is the reason why only 22.5% of detected proteins (7 out 31) are characteristic of SEVs, according to Vesiclepedia (http://www.microvesicles.org/).
8.2 RNA Characterization
[0293] RNA sequencing technique was used to identify the small RNA content of SEVs compositions. The RNA was isolated using EXIQON miRCURY™ Isolation kit (Cell & Plants) and small RNA quality and quantification was performed in Bioanalyzer 2100. 1.5 ng of small RNA was used for small library construction according to Ion Total RNA-Seq Kit v2 protocol. The pooled libraries further processed on Ion Chef™ System and the resulting 530-chip was sequenced on Ion S5™ System. Cufflinks tool was used for annotation and estimation of the relative abundance of each gene in each sample.
[0294] SEVs isolated with the process of the present invention (INV) have different species of small RNAs, with transference RNA being the most abundant RNA species, representing in average 57%. microRNAs (miR) are the second most predominant specie (˜27%) followed by ribosomal RNA (˜6%) and other minor categories. Among samples, there are no major differences in RNA species. Again, although more samples are needed to test, a range of miR can be defined as shown in Table 4 and Table 5.
8.3. Lipid Characterization
[0295]
TABLE-US-00007 Percentage of Lipid Total Lipids (%) CE 0.05 ± 0.05 DAG 1.3 ± 0.4 PA 3.18 ± 0.6 PC 30.9 ± 3.3 PE 13.0 ± 0.5 PG 0.25 ± 0.1 PI 3.8 ± 0.3 PS 29.4 ± 3.7 SM 18.0 ± 1.6 TAG 0.074 ± 0.13 Total 100
Example 9. Comparative Tests
[0296] For comparison purposes between the performance of a standard process of SEVs isolation as disclosed in WO2017163132 (COMP comparative process) and the process of the present invention (INV inventive process), Small Extracellular Vesicles from several equal samples of UCB were isolated and analysed being the results presented in the tables below.
[0297] The process of the present invention was performed according to what is described in the examples 1 to 8.
Under Ischemia Conditions
[0298] Before ischemia, the characterization of the cells derived from automatic UCB processing contain higher amounts of RBCs than the samples processed manually. Nevertheless, the ratio lymphocyte/monocyte in each group was maintained, and is not statistically different between the two UCB processing methods. More importantly the automatic UCB processing samples have more viable cells as shown by its lower percentage of cell death (˜2%) when compared to manual processing (˜8%).
[0299] After the Ischemia protocol (18h, 37° C. under 0.5% O2 and 5% CO.sub.2) each population maintained their representativeness namely the lymphocyte/monocyte ratio was maintained. As expected, due to the extreme conditions the cell death increases in both groups.
[0300] Table 7 shows the comparison between proteins isolated by the COMP process and INV process, and Table 8 presents the comparison of isolated proteins by both processes grouped by their biological functions.
[0301] Further, the percentage of small RNA species in Small Extracellular Vesicles isolated by the COMP and INV processes was also evaluated and is shown in Table 9, whereas Table 10 lists microRNAs identified in Small Extracellular Vesicles isolated by the INV process and COMP process.
TABLE-US-00008 TABLE 7 Comparison between proteins isolated by the COMP process and INV process COMPARATIVE UniProt UniProt ID Name ID Name ID Name Q96PDS PGLYRP2 P60953 CDC42 P04080 CSTB Q9UIA9 XPO7 Q15758 SLC1A5 P02656 APOC3 P05090 APOD P59998 ARPC4 P01008 SERPINC1 Q86UX7 FERMT3 Q9HDC9 APMAP O43760 SYNGR2 P01011 SERPINA3 P62834 RAP1A O15243 LEPROT P05156 CFI P08865 RPSA O15230 LAMA5 P01019 AGT Q99623 PHB2 Q8IUA7 ABCA9 P05107 ITGB2 O75340 PDCD6 P61803 DAD1 P02652 APOA2 Q680K9 HBM Q9NZT1 CALML5 P22792 CPN2 P13639 EEF2 P26641 EEF1G O00560 SDCBP P54802 NAGLLI P48735 IDH2 P26038 MSN P32942 ICAM3 P07437 TUBB Q99880 HIST1H2BL P16070 CD44 P04433 KV311 P08575 PTPRC Q8NG11 TSPAN14 P00739 HPR P04899 GNA12 P35613 BSG P08631 HCK Q8WUM4 PDCD6IP O75131 CPNE3 P24158 PRTN3 P51149 RAB7A Q6UXB8 PI16 Q96897 MYADM P0C0L4 C4A. C4B Q95365 1B38 B0I1T2 MYO1G P11215 ITGAM P02743 APC5 Q9Y336 SIGLEC9 P01861 IGHG4 Q90GMS FETUB P51884 LUM P05543 SERPINA7 O15162 PLSCR1 Q66K66 TMEM198 P04843 RPN1 P20702 ITGAX CM3759 SYNGR1 P0DOY3 IGLC3 P0DMV9 H5PA1A P02794 FTH1 P01009 SERPINA1 P37837 TALDO1 P04004 VTN Q9BQE3 TUBA1C P49368 CCT3 Q9Y286 SIGLEC7 P62873 GNB1 P12955 PEPD Q14739 LBR P09525 ANXA4 P14625 HSP90B1 P61981 YWHAG P30740 SERPINB1 P30101 PDIA3 P06276 BCHE Q92930 RAB88 P07339 CTSD Q76L83 ASXL2 COMMON INVENTIVE UniProt UniProt UniProt ID Name ID Name ID Name P0DOX2 IGA2 P46976 GYG1 P16157 ANK1 P0DOX7 IGK P69905 HBA1 P0C0L5 C4B P01871 IGHM P68871 HBB P31944 CASP14 P01619 KV320 P02042 HBD Q16778 HI5T2H2BE Q5VTE0 EP1A3 P69891 HBG1 P01859 IGHG2 P01876 IGHA1 P69892 HBG2 Q05707 COL14A1 P04217 A1BG P02008 HBZ Q01518 CAP1 P01023 A2M P62805 HIST4H4 P02549 5PTA1 P63261 ACTG1 P00738 HP P16403 HIST1H1C P12814 ACTN1 P02790 HPX P00736 C1B P43652 AFM Q86YZ3 HRNR P0DOY2 IGLC2 P02765 AHSG P11142 HSPA8 P12273 PIP P02768 ALB P08514 ITGA2B Q96P63 SERPINB12 P04075 ALDOA P05106 ITGB3 P07478 PRSS2 P02760 AMBP P01591 ICHAIN P02649 APOE P04083 ANXA1 P14923 IUP Q14254 FLOT2 P50995 ANXA11 P01042 KNG1 Q08188 TGM3 P07355 ANXA2 P11279 LAMP1 P67936 TPM4 P12429 ANXA3 P31025 LCN1 Q06830 PRDX1 P08758 ANXA5 P07195 LDHB O43866 CDSL P08133 ANXA6 Q08380 LGALS3BP P08238 HSP90AB1 P20073 ANXA7 P02750 LRG1 Q9NZD4 AHSP O75882 ATRN P02788 LTF P01768 HV330 P25311 AZGP1 P61626 LYZ Q71D13 H15T2H3C P30043 BLVRB Q08431 MFGE8 P40926 MDH2 P02745 C1QA P05164 MPO P19338 NCL P02746 C1QB P35579 MYH9 Q93077 HIST1H2AC 502747 C1QC P60660 MYL6 P01031 C5 P09871 C1S Q6UX06 OLFM4 P20618 PSMB1 Comparative: Proteins isolated by the COMP process Common: Proteins isolated by both COMP and INV processes Inventive: Proteins isolated by the INV process
TABLE-US-00009 TABLE 8 Comparison of isolated proteins by INV and COMP processes grouped by their biological functions INV COMP % % Biological function proteins p-value proteins p-value neutrophil 20.37 P = 0.004 25.17 P < 0.001 degranulation cell adhesion 1.85 P = 0.72 9.79 P < 0.001 extracellular matrix 1.85 P = 0.41 8.39 p = 0.001 organization leukocyte migration 7.41 p = 1 7.69 p = 0.012 cellular protein 3.7 p = 1 7.69 p = 0.009 metabolic process innate immune 9.26 p = 1 6.99 P = 1 response regulation of 12.96 P < 0.001 6.29 p = 0.004 complement activation platelet 5.56 p = 1 6.29 p = 0.014 degranulation Fc-gamma receptor 9.26 P = 0.0796 5.59 p = 0.217 signaling pathway involved in phagocytosis receptor-mediated 9.26 p = 1 5.59 p = 0.541 endocytosis inflammatory response 7.41 p = 1 4.9 P = 1 complement activation, 12.96 P = 0.002 3.5 P = 1 classical pathway complement activation 11.11 P = 0.005 3.5 P = 1 immune response 7.41 p = 1 3.5 P = 1 protein 12.96 P = 0.187 2.1 P = 1 deubiquitination Fc-epsilon receptor 7.41 p = 1 1.4 P = 1 signaling pathway keratinization 5.56 p = 1 0.7 P = 1 MAPK cascade 3.7 p = 1 0.7 P = 1 NK-kB siganaling 10.71 P < 0.001 0.68 p = 0.37 T cell receptor 10.71 P < 0.001 1.37 p = 0.33 signalling pathway
TABLE-US-00010 TABLE 9 Percentage of small RNA species in Small Extracellular Vesicles isolated by the COM process and by the INV process. INV COMP RNA Average SD Average SD snRNA 1.99 1.08 0.58 0.10 snoRNA 1.47 0.98 0.41 0.09 rRNA 6.19 0.97 14.40 1.67 microRNA 26.47 5.40 8.40 1.02 tRNA 57.23 6.87 6.25 2.61 other 6.65 1.23 69.26 4.18
TABLE-US-00011 TABLE 10 List of the microRNA identified in Small Extracellulare Vesicles isolated by the INV process and the COMP process Common INV exclusive hsa-let-7a-5p hsa-miR-16-5p hsa-miR-26b-5p hsa-let-7b-5p hsa-miR-22-3p hsa-let-7f-5p hsa-miR-17-5p hsa-miR-29b-3p hsa-let-7c-5p hsa-miR-26a-5p hsa-let-7g-5p hsa-miR-181a-5p hsa-miR-30d-5p hsa-let-7i-5p hsa-miR-27a-3p hsa-miR-103a-3p hsa-miR-191-5p hsa-miR-451a hsa-miR-130a-3p hsa-miR-27b-3p hsa-miR-106b-5p hsa-miR-19a-3p hsa-miR-142-3p hsa-miR-144-3p hsa-miR-30b-5p hsa-miR-142-3p hsa-miR-19b-3p hsa-miR-146b-5p hsa-miR-144-5p hsa-miR-3184-3p hsa-miR-146a-5p hsa-miR-20a-5p hsa-miR-29a-3p hsa-miR-185-5p hsa-miR-376c-3p hsa-miR-150-5p hsa-miR-21-5p hsa-miR-101-3p hsa-miR-18a-5p hsa-miR-486-5p hsa-miR-15a-5p hsa-miR-223-3p hsa-miR-205-5p hsa-miR-92a-3p hsa-miR-15b-5p hsa-miR-23a-3p hsa-miR-221-3p hsa-miR-93-5p
[0302] In terms of Small Extracellular Vesicles yield (
[0303] In summary, with this new process it is possible to increase the quantity of Small Extracellular Vesicles isolated from the conditioned media and this is very important in the production of SEVs.
[0304] Further, with this GMP-compliant optimized methodology it also possible to produce SEVs with very similar biophysical properties when compared to the prior art process (COMP).
Example 10. SEVs Effect in Immune Cells
10.1 PBMNCs
Differentiation of Regulatory T Cells
[0305] Healthy blood donors PBMNCs were incubated with SEVs compositions (1×10.sup.10 part/mL) or the vehicle for 6 days. Cells were washed and stained for FACS analysis. Results are presented as mean±SD (n=5).
[0306] Statistical analysis was performed using student's t-test. To assess the immunomodulatory potential of the SEVs compositions, tests were performed on healthy human peripheral blood mononuclear cells (PBMNCs) with or without the presence of SEVs compositions of the invention and T cell polarization was evaluated.
[0307] After one application of SEVs composition and after 6 days of incubation, the prevalence of Treg (positive for CD25 and FoxP3) within T cell subpopulations was evaluated. SEVs composition was able to significantly increase the percentage of Treg in culture (9.68±1.89% in treated, against 5.24±2.53% in control) (
Differentially Influence mRNA Expression of Lymphocytes
[0308] Additionally, T helper cell polarization was evaluated by quantifying the expression of lineage genes in the lymphocyte subset through time. Master transcription factors was chosen to evaluate each lineage: GATA3 for Th2; T-bet for Th1; Foxp3 for Treg; and RORYT for Th17.
[0309] To complement the analysis, the expression of IFN-Y for CD8 T cells was also evaluated.
[0310] After 24h of incubation with SEVs, there are no significant differences in the transcription factors analyzed. After 48h, the differences started to accentuate between treated and non-treated groups, and at day 6 the transcriptional profile indicates a differentiation to the Treg phenotype, with increased expression of FoxP3 and TGF-β1.
[0311] SEVs does not seem to impact Th1 and Th2 T cell subset, as observed from the mRNA levels of T-bet and Gata3. Interestingly, it was observed a significant inhibition of RORYT, which suggests that there is a decreased expansion of Th17 cells upon SEVs treatment (
[0312] Previous reports showed that approximately 50% of activated T cells expressing this transcription factor produce IL-17A, a key cytokine in the psoriasis pathophysiology.
[0313] Based on these results, we hypothesize that exposing T-lymphocytes to SEVs will increase the population of Treg cells while limiting the Th17 T-cell subsets.
[0314] These results are corroborated by the POC done in a mice model of psoriasis.
[0315] Moreover, it is well recognized that IFN-Y plays a critical role in the control of CD8 T cell expansion and contraction during immune responses. This T cell subset is responsible for the release of IFNg and TNFa, cytotoxic granules and also for destroying infected cells. SEVs induces downregulation of IFNg expression which suggests a lower number CD8+ T cell and a consequent anti-inflammatory effect (
Differentially Influence FoxP3 mRNA Expression in PBMNCs
[0316] Healthy blood donors PBMNCs were incubated with SEVs (1×10.sup.10 part/mL) or the vehicle for 6 days. Total RNA was extracted, and gene expression analysis was performed using S-actin as reference gene. The normalized expression levels were calculated based on 2.sup.−ΔΔCt. Statistical analysis was performed using one-way ANOVA (n=3).
[0317] A different strategy was used to analyze T cell polarization when treated with SEVs. Total RNA was extracted from the total PBMNCs mixture instead of lymphocyte subset. Some of the previous differences were diluted maybe be due to an increase of heterogeneity of mRNA. Nevertheless, the increase of FoxP3 transcriptional factor expression was again observed. The immune modulatory effect of SEVs indicates toward an enrichment of Treg in the microenvironment, potentiating the immune tolerance. The other analyzed factors were not significantly altered by SEVs, with exception of Gata3 expression. Although not exclusively, Gata3 activity regulates Th2 differentiation and cytokine secretion. This decrease can be particularly important in in the treatment of asthma and allergic diseases.
Impact of miR-150-5p and miR-146-5p on Lymphocytes Expression Profile After 48h
[0318] Healthy blood donors PBMNCs were transfected with both miR using Viromer Blue. 2 days after transfection, lymphocyte compartment was sorted based on morphological features (FSC and SSC) and total RNA was extracted. Gene expression analysis was performed using β-actin as reference gene. The normalized expression levels were calculated based on 2-ΔΔCt. Statistical analysis was performed using student's t-test (n=3).
Impact of miR-150-5p and miR-146-5p on Lymphocytes Phenotype 6 Days after Transfection.
[0319] Healthy blood donors PBMNCs were transfected with both miR using Viromer Blue. 6 days after transfection, the percentage of Treg (CD4.sup.+CD25.sup.+FoxP3.sup.+) and Th17 (CD4.sup.+IL17A.sup.+IFN.sup.−) was analyzed by flow cytometry. Statistical analysis was performed using One way ANOVA (n=3).
[0320] At this point, we had some evidences that SEVs has immunotolerant properties namely through T cell differentiation towards a regulatory phenotype. To further understand the mechanism of action of our biological product, two different microRNAs (miR) that are present in SEVs were directly transfected into PBMNCs mixture. miR-146-5p is highly expressed in regulatory T cells. It is indispensable for their immunosuppressive function such as its deletion resulted in a breakdown of immunological tolerance manifested in a IFNγ-dependent immune-mediated lesions in a variety of organs. miR-150-5p is reported as Treg differentiation driver in conjugation of other miRs although its role is demonstrated through induction and proliferation of CD8.sup.+ effector cells.
[0321] Regarding miR-146a-5p, our results showed a significant increase FoxP3 expression (
[0322] Interestingly, miR-150-5p transfection resulted in an overexpression of RORYT and FoxP3, which suggests that this miR may induce the differentiation into a new regulatory T cell subtype that was recently described as T regulatory 17 FoxP3.sup.+RORYt.sup.+, which is able to suppress Th17 differentiation and control autoimmunity.
[0323] In parallel with gene expression analysis, flow cytometry data also showed an increase in regulatory T cell subset in PBMNCs transfected with both miR (
[0324] These results indicate that a possible enrichment with these miRs could potentiate immunomodulatory potential of SEVs, generating an improved version of the product.
Example 11. Effect in Psoriasis—In Vitro
11.1. Imiquimod-Induced Psoriatic Model
[0325] SEVs decreases TNF-a expression while potentiate VEGFA and TGFb1 Expression in Psoriatic Like-NHEK Model
[0326] NHEK were terminally differentiated for 3 days with 2 mM of CaCl.sub.2. Then, IMQ was added for 3h in order to potentiate their pro-inflammatory phenotype. Finally, treatment was administrated in co-incubation with IMQ for 3h and cells were collected for RNA extraction. 500 ng of RNA was used for reverse transcription reaction. mRNA expression was normalized by b-actin expression gene (n=3 for each condition).
[0327] Based on previous results that demonstrated that SEVs is able to not only increase the number of Treg but also decrease the expression of RORYT, a master transcriptional factor of Th17 T cell lineage, we decided to study the impact of SEVs in models of psoriatic epidermis.
[0328] For that, first we differentiated keratinocytes with Ca.sup.2+ to mimic the epidermis outermost layer and applied 5% of IMQ, a compound described to resemble most of the psoriasis phenotype, through its proinflammatory properties.
[0329] After 3h of simultaneous incubation (SEVs and IMQ), we observed decreased expression of TNF-α and an increased expression of VEGF-a and TGF-b1 in response to SEVs (
[0330] TNF-α is a pro-inflammatory cytokine that amplifies inflammation through several distinct pathways: facilitating entry of inflammatory cells into lesioned skin through induction of adhesion molecules on vascular endothelial cells; stimulating keratinocyte production of other pro-inflammatory mediators and activating dermal macrophages and dendritic cells. Thus, its inhibition promoted by SEVs could ameliorate psoriasis pathophysiology by blocking the vicious activating cycle of dendritic cells.
[0331] On the other hand, SEVs stimulated VEGFA and TGFb1 expression. VEGFA plays a key role in the establishment of an immunosuppressive tumor microenvironment, as it inhibits the maturation of dendritic cells, and stimulates the proliferation of Tregs.
[0332] Also, TGF-b1 has been shown to limit inflammation and to control peripheral tolerance by both direct and indirect regulation of autoreactive T cells. Although TGF-β is essential for T cell tolerance, it has recently been proposed that loss of TGF-β signaling alone is insufficient to induce autoimmunity.
Proinflammatory Cytokine Expression in Psoriatic Like-NHEK after SEVs Treatment for 3 Days
[0333] NHEK were terminally differentiated for 6h with 2 mM of CaCl2). Then, CaCl and IMQ were added for 18h in order to potentiate their pro-inflammatory phenotype and differentiation. Finally, treatment with SEVs (1×10.sup.10 part/mL) was administrated in co-incubation with IMQ for 3 days. Cells were collected for RNA extraction and supernant was also collected for ELISA. 1 μg of RNA was used for reverse transcription reaction. mRNA expression was normalized by b-actin expression gene (n=3 for each condition).
[0334] In a similar model, SEVs stimulus potentiated the downregulation of IL6 expression when compared to the vehicle (
11.2. Psoriasis-Like Epidermis
[0335] Gene Expression Profile in a Three-Dimensional Epidermis-Like Psoriatic Model after SEVs Treatment for 5 Days
[0336] RNA was extracted using RNAeasy Mini Kit (Qiagen) according to manufacture recommendations. 500 ng of RNA was used for reverse transcription reaction. mRNA expression was normalized by b-actin expression gene (n=3 for each condition).
[0337] To confirm the potential anti-psoriasis effect in skin, we have used a commercial three-dimensional epidermis model (PSORIASIS LIKE EPIDERMIS, Sterlab). The model was treated for 5 days with a single dose of SEVs every day. At the end of experiment, cells were collected for RNA analysis and supernatant for protein secretion quantification.
[0338] SEVs downregulates the mRNA expression of several cytokines such as IL-6, IL-8, TNF-a and IFN-g (
[0339] Additionally, we have analyzed the expression of antimicrobial peptides (S100A7 and DEFB4) and found consistent downregulation of both (
[0340] Furthermore, these antimicrobial peptides contribute to chemotaxis of neutrophils and T cells, exacerbating psoriasis phenotype. Decreasing the expression of these peptides may significantly contribute to ameliorate the psoriatic pathophysiology.
[0341] Moreover, SEVs does not seem to impact the differentiation stage of keratinocytes as no differences were seen on KRT5. Interestingly, Cox-2 expression is downregulated in SEVs treated group, which is expected to contribute for the control of inflammation. In turn, this enzyme is downregulated by miR-16-5p (Shanmugam, Reddy, and Natarajan 2008), which is one of the most abundant microRNA species in SEVs, suggesting that the mechanism of action may be miR-16-dependent.
[0342] Protein secretion in a three-dimensional epidermis-like psoriatic model after SEVs treatment for 5 days. After 5 days of culture, the supernatant was collected and analyzed by ELISA. (n=3 for each condition). When analyzing protein secretion we found that SEVs decreases CCL20 and TNF-α (
Example 12. Effect in Psoriasis—In Vivo
Topic-SEVs Amelirorates Psoriasis-Like Skin Inflammation in Mice.
[0343] Photographs of the shaved skin on mice's backs were taken 6 days after topical treatment. Clinical scoring was performed by ranking severity of erythema (redness from 0 to 4), affected area (0-3) and desquamation (scale from 0-2). Infiltration of IMQ-treated back skin by inflammatory cells was revealed by flow cytometry using, granulocyte (Ly6G) (B) and TCRab+(C) markers. Lymph node T cell phenotyping was performed by flow cytometry using CD4, CD8 and GL3 T cell markers as well as CD44 and CD62L (memory associated marker) and CCR6, Th17 marker. Data represent means±SEM for n=6 mice per group. P-values were calculated by Student's t-test.
[0344] To access the efficacy of SEVs on a psoriatic in vivo model, IMQ-induced skin inflammation was chosen as a model that phenotipically resembles psoriasis in mice. The model was induced by topical application of Aldara, a cream preparation containing 5% IMQ, every day. IMQ is a ligand for the toll-like receptors TLR7 and TLR8 that exacerbates psoriasis at both the local treated areas as well as distant sites. TLR7 and TLR8 are expressed by monocytes, macrophages and dendritic cells that when are activated lead to pro-inflammatory cytokines and chemokines production. Application of Aldara on hair-free back of mice results in the development of psoriasis-like lesions within 5 days of application and is underpinned by an influx of various cells as well as hyperplasia of the epidermis.
[0345] During the six days of consecutive treatment with SEVs topically, the mice skin clinical score was monitored. The final value resulted in a sum of three parameters: severity of erythema (redness from 0 to 4), affected area (0-3) and desquamation (scale from 0-2). Topic application of SEVs delayed the appearing of psoriatic characteristics and attenuated them over time (
[0346] Additionally, topical treatment with SEVs significantly decrease the number of skin infiltrating neutrophils (CD45.sup.+CD11b.sup.+Ly6G.sup.+CD64.sup.−) and mature T lymphocytes (CD45.sup.+CD3.sup.+TCRαβ.sup.+) (
[0347] Moreover, it is generally accepted that the pathogenesis of psoriasis involves accumulation of effector T-cells within lymph nodes and their subsequent migration into the skin through the blood system. In our experiment, we observed that Th17 cells and Υδ expressing cells were significantly decrease in the mice lymph nodes treated with SEVs (
[0348] We believed that the decrease in IL-17 producing cells in lymph nodes would decrease further infiltration in mice skin and attenuate psoriasis. This effect will be also potentiated by the decrease of neutrophil number that will diminish the quantity of cytokines/chemokines in the microenvironment and hamper the recruitment of more T helper cells.
Example 13. Up-Scaling the Process of the Present Invention
[0349] The purification of SEVs was tested in conditions that mimic the ones used in a GMP unit with the use of Tangential Field flow (TFF) for micro and ultrafiltration (UF). The results were compared to our lab-scale UF/SEC method. The idea was to observe by biophysical characterization of the SEVs if the TFF could be used in the future as an SEVs isolation methodology.
Example 14: SEVs Promote In Vivo Wound Healing
[0350] We decided to validate if the inventive process of isolation of SEVs would have an impact in their bioactivity and potential to treat skin wounds.
[0351] Briefly two excisional wounds were performed in the dorsal region STZ-induced diabetic mouse model, and applied SEVs in solution (2.5×10{circumflex over ( )}8 particles) topically, twice a day. The wound area was measured during the experiment in order to assess the efficacy of SEVs in the wound healing kinetics. The results show that the SEVs isolated with the prior art method (UC—ultracentrifugation) are less bioactive than the ones isolated with inventive method UF-SEC (
REFERENCES
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